DNA encoding an antigenic protein derived from Eimeria tenella and vaccines for prevention of coccidiosis caused by Eimeria tenella

ABSTRACT

Nucleic acid molecules are provided which encode antigenic proteins capable of inducing in a chicken an immune response conferring protection against Eimeria tenella. Expression vectors containing the nucleic acid molecules are also provided. Methods for producing the proteins or antigenic polypeptides having amino acid sequences included within these proteins are also provided.

This is a continuation of U.S. Ser. No. 125,012, filed Nov. 24, 1987,now abandoned, which is a continuation-in-part of U.S. Ser. No. 805,824,filed Dec. 6, 1985, now U.S. Pat No. 4,874,705 which is acontinuation-in-part of U.S. Ser. No. 734,085, filed May 16, 1985, whichis a continuation-in-part of U.S. Ser. No. 617,483, filed Jun. 5, 1984,now abandoned, the contents of all of which are hereby incorporated byreference into the present application.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byArabic numerals within parentheses. Full citations for these referencesmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

The phylum Apicomplexa includes hundreds of different organismsbelonging to the order Eucoccidiorida. The genus Eimeria is includedwithin the order of true coccidian agents. Of the organisms belonging tothis genus, several species are of recognized importance to the chickenindustry. These species include Eimeria tenella, E. maxima, E.acervulina, E. necatrix, E. brunetti, E. mivati, E. mitis and E.praecox.

Differentiation of species is based on the site of infection within thehost and oocyst morphology. To date, biochemical markers have not beenused for speciation, although differences have been noted for each ofthe above species.

For avian Eimeria, the entire life cycle is completed within a singlehost. The life cycle is complex consisting of asexual and sexual stagesdepending upon the Eimeria species involved. The infective stage is thesporulated oocyst. Upon being ingested in contaminated feces, food orwater, sporulated oocysts excyst within the digestive tract as a resultof the combined action of mechanical shearing and enzymatic hydrolysisof the sporocyst cap. The liberated sporozoites traverse epithelialcells within specific regions of the intestine.

Development begins within the Crypt of Lieberkuhn to the level of firstgeneration meronts; the meront is a transitional stage consisting ofrounded organisms with a more pronounced nucleus, plus increased energygenerating and protein synthesizing capacity. Development offirst-generation merozoites follows due to multiple fission of meronts.The release of first-generation merozoites destroys the host cell, andthe parasites migrate to infect new host cells undergoing a secondasexual cycle. Meronts develop to the level of second-generationmerozoites destroying additional epithelial cells as they are released.Further destruction of host cells follows with the liberation of thethird-generation merozoites. The number of merozoite generations variesfrom one Eimeria species to another.

Sexual development commences with the production of microgametes andmacrogametes through the process of gametogenesis. Liberatedmicrogametes fertilize macrogametes to form zygotes. Development ofimmature oocysts is followed by rupture of the host cell. Oocysts,released into the lumen of the gut, are passed through the feces to theenvironment and mature (sporulate) in the presence of atmosphericoxygen.

The process of parasite development is self-limiting if the host ingestsno additional oocysts. However, this proves to be an unrealisticexpectation in crowded poultry houses.

Disease due to Eimeria can result in severe economic losses associatedwith diminished feed efficiency and pathologic manifestations.

The pathology of coccidiosis due to E. tenella and E. necatrix is inlarge part related to the rupture of host cells during the release ofmerozoites, while host cell rupture during the release of E. maximaoocysts contributes to the pathology seen with that species. Bleedingwithin the gut is related to rupture of small capillaries servicing theepithelium. It may be difficult to control the progress of disease usingcoccidiostats, once asexual development is established. Secondaryinfection often complicates the disease caused by Eimeria. Death canocccur within 4-7 days in infected birds infected with E. tenella or E.necatrix. However, death rarely occurs as a result of infection by E.maxima.

A consistent property of the coccidia is that the sporozoites initiatethe infection process within very specific tissue sites (39, 45, 57).The site specificity of infection is a characteristic commonly used forspeciation of Eimeria. For example, the asexual stages of E. necatrixshow a propensity for invasion of epithelial cells residing within themid-intestine, while sexual stages develop primarily in the cecalpouches.

Much of the work on immunity to coccidiosis has been confined to humoralimmunity, more specifically to serum antibodies. Studies have shown alack of correlation between serum antibody and resistance to disease(59). However, most available data support the contention that a localresponse with involvement of the secretory immune system or cellmediated immunity (CMI), or both, are involved in the protectiveresponse.

Interference with recognition, penetration and/or attachment ofpathogens to host cells has a demonstrated protective effect as shownwith viral, bacterial and protozoan models. Genetic deletion of key hostcell receptors or pathogen attachment features can prevent the initialcolonization process (16, 54). Alternatively, secretory antibodies caninterfere with the colonization process by binding to, and consequentlymasking requisite receptors (32, 74). More than one immunoglobulin classhas been reported to have the capacity of interfering with the initialcolonization process of Eimeria tenella (13). However, recent reportsindicate that only production of secretory IgA has been correlated withnatural protective immunity (12, 59). Porter and Davis (13) and others(59) reported that secretory IgA neutralizes the extracellular stages ofthe parasite either by significantly limiting penetration or sodebilitating those organisms which did penetrate as to preventsubsequent development.

It has been estimated that an amount approaching $0.5-1.0 billion isspent annually by producers worldwide to combat disease, or to curb thedevastating effect of coccidiosis in chickens (39, 52). Even withcontrol measures currently in use, poultry losses are substantial withestimates in the multi-million dollar range (77).

Currently, the most widely used means of controlling Eimeria in chickensis through the application of antiprotozoal chemical feed additives. Thespecific composition varies with the coccidiostat used, and each productaffects only certain stages of the coccidian life cycle (39, 51, 58).Disadvantages of using coccidiostats are many, including short-termresidual protection in birds, occasional diminished performance,invocation of resistance to the drug in parasites, and to some extent,safety. Products currently remain on the market for only a few yearsbecause of the development of drug resistant strains. This addsconsiderable pressure on the cost of development and continuedmanufacture of efficacious products (51).

Protection of birds by immunization has met with some success.Investigators have been able to invoke limited protection usingpreparations of killed organisms (1, 41, 43). A more effective approachfor immunization of chickens has been with the use of a live protozoalproduct--e.g. Coccivac™ (15). The product, being a multivalentcomposition containing low doses of viable oocysts, is administered indrinking water to invoke a mild parasitemia in birds. A drawback of thisproduct has been occasional depressed performance of birds during thefirst weeks following administration. Variables such as excessive dosingor moisture content of bedding have even led to severe outbreaks ofcoccidiosis. See also, U.S. Pat. No. 3,147,186 (1964) which concerns theuse of viable, sporulated oocysts of E. tenella to immunize chickens andU.S. Pat. No. 4,301,148 (1981) which concerns the use of sporozoites ofE. tenella for the same purpose.

An alternative means of introducing the live vaccine into broiler housesis by way of the feed. This has been considered in a recent Britishpatent (GB2,008,404A). Prior to mixing with the feed, fully virulentoocysts of E. tenella are encapsulated in a water soluble polysaccharideto protect against desiccation. The oocysts are in sufficient amountsonly to induce subclinical infection. Though the immunizing ability wasfound to be excellent, no development of this method is foreseen due toquestionable field acceptability. However, if attenuated strains of allthe important coccidia could be developed, the procedure may be moreacceptable.

Efforts have indeed been made to develop Eimeria lines of reducedvirulence. Some species have been successfully attenuated throughchicken embryo passage (19, 37, 40, 66). These strains have diminishedability to cause disease, yet have retained sufficient immunogenicity toinvoke immunity. Some problems do, however, remain with the handling ofthese strains. As examples, the attenuated variants of E. necatrix havea critical passage limit whereby more or less embryo passage can resultin loss of immunogenicity or maintenance of the original virulent form.Furthermore, some attenuated organisms revert to the virulent form uponminimal back-passage through chickens (38, 68). Thus, problemsassociated with maintaining consistent properties in attenuatedorganisms are apparent.

Attenuation by precocious selection has also been practiced when Eimeriastrains cannot be readily passaged through embryonated eggs. In thisprocess, shed oocysts are harvested late in the prepatent period priorto the onset of heavy oocysts shedding (28, 48, 50, 67). Such selectionresults in cultures having abbreviated life cycles, and a correspondingdiminution in virulence properties (28, 48, 50, 67). Though the trait ofprecocity for E. tenella (29) and E. acervulina (49) has beendemonstrated to be genetically stable, not enough information is knownabout this method to assess its usefulness as a tool in the poultryindustry.

There is little information available about the surface antigencomposition of avian coccidia. Hybridoma cell lines which secretemonoclonal antibodies directed to antigens on the surface of sporozoitesof Eimeria tenella have been reported (82). The antigens were notidentified, other than that their molecular weights were between 13 and150 kilodaltons. Additionally, no biological significance or describedefficacy in a vaccine was attributed to the antigens. European PatentPublication No. 135,712 also discloses monoclonal antibodies which reactwith sporozoites of E. tenella. E. tenella sporozoite antigens aredisclosed by this publication. Furthermore, European Patent PublicationNo. 135,073, corresponding to U.S. Pat. No. 4,650,676, disclosesmonoclonal antibodies which react specifically against merozoites andsporozoites of E. tenella. Merozoite antigens derived from E. tenellaare described.

Previous work in the laboratory of M. H. Wisher suggests the presence ofapproximately 16 polypeptides identified by surface iodination ofexcysted sporozoites of E. tenella and having molecular weights form20,000 to greater than 200,000 (81). Additionally, European PatentPublication No. 167,443 discloses extracts from sporozoitesor sporulatedoocysts of E. tenella which may be used as vaccines to protect againstcoccidiosis. These extracts contain a plurality of polypeptides, one ormore of which may be used as an antigen to protect against coccidiosis.Moreover, International Publication No. WO/00528 discloses a cloned geneor fragment thereof from E. tenella which encodes antigenic proteins.These proteins bind with a monoclonal or polyvalent antibody directedagainst an antigenic protein of avian coccidia.

Subunit approaches to vaccine development have proven successful overthe past few years. In such approaches, candidate protective antigensare identified and characterized for the purpose of eventual preparationon a large scale. In studying parasite antigens, one research group usedmonoclonal antibodies to identify a potential protective antigen on thesurface of Babesia bovis (83). A B. bovis antigen of 44,000 daltons hasbeen identified, which when purified and injected into experimentalanimals afforded some level of protection against primary challenge. Animmunologically important 30,000 dalton protein of Toxoplasma gondii hasalso been identified using monoclonal antibodies (31).

Since mid-1981, Danforth and coworkers have published several papers inwhich they indicate the possibility of producing monoclonal antibodiestoward antigens of avian Eimeria species (9, 10, 11). Similarly, Speer,et al. (69, 70) have demonstrated the development of hybridomas againstE. tenella and some physiologic properties thereof. Antibody-secretinghybridomas have been selected on the basis of an indirect fluorescentantibody test (10). The patterns of reaction, as observed withultraviolet microscopy, have varied depending upon the monoclonalantibody used. Patterns have included exclusive reaction withsporozoites only vs reaction with sporozoites and merozoites; stainingof the anterior portion of the sporozoite vs the entire membrane; andstaining of distinct internal organelles vs non-descript internalstaining (11).

Although the preparation of murine-origin hybridomas producingmonoclonal antibodies is commonly practiced by those familiar with theart, there is nothing to suggest that the direct and specific selectionof sporozoite-neutralizing hybridomas against the species E. tenella andE. necatrix or merozoite-neutralizing hybridomas against the species E.maxima will subsequently identify virulence determinants of thesespecies which may be useful in the development of a subunit vaccine.

This invention concerns the identification, characterization,preparation and use of polypeptide antigens for development of immunityto coccidiosis caused by E. tenella, E. necatrix and E. maxima.Recombinant polypeptide antigens, including fusion proteins, are alsodescribed.

The antigens are capable of being precisely dispensed interms of directantigenic content and cannot cause disease thus avoiding vaccinestrain-related outbreaks and reversions or changes in immunologicproperties.

Due to the large economic losses caused by coccidiosis in chickens,vaccines against E. tenella, E. necatrix and E. maxima are desirable.Using hybridoma technology, applicants have identified and purifiedpotential protective antigens for use in subunit vaccines. Use of such asubunit vaccine avoids vaccine strain-related outbreaks and reversionsor changes in immunological properties associated with the use of a livevaccine.

The quantity of parasite antigens that can be prepared from the organismis quite low and very costly. Recombinant DNA cloning and expressiontechniques have opened up a new approach to producing large amounts ofprotective antigens inexpensively. In simplest terms, these techniquesrequire that DNA sequences encoding all or part of the antigen be placedin a cell, under the control of the genetic information necessary toproduce the antigenic protein in that cell. The genetic information maybe synthetic DNA (17), genomic (e.g., viral) or chromosomal DNA, or cDNAmade from the mRNA encoding the antigen. The latter approach is the mostdirect method for complex organisms such as Eimeria sp.

However, because the cDNA only contains genetic informationcorresponding to the amino acid sequence of the antigen, it must beinserted into expression vectors that provide the genetic signalsnecessary for expression of the cDNA gene (i.e., transcription andtranslation). The antigens can be synthesized either alone or asproducts fused to another protein in E. coli.

Production of an effective subunit vaccine in E. coli has been reportedfor foot and mouth disease virus of swine and cattle (33, 66). Foot andmouth disease virus surface antigens were produced as fusion proteinantigens in E. coli. Significant levels of virus-neutralizing antibodywere raised when cattle and swine were immunized with these antigens.The recombinant DNA-derived antigens gave protection against challengewith foot and mouth disease virus.

In contrast to simple organisms such as foot and mouth disease viruswhere the genome and surface proteins have been studied extensively,very little is known about the molecular biology of Eimeria. Wang andStotish (79, 80) reported rapid but transient RNA and protein synthesisin E. tenella during the first 6-8 hours after initiation of sporulationand suggested that all protein and nucleic acid synthesis duringsporulation occurs in these first few hours. For example, Stotish et al.(72) reported a 30,000 dalton glycoprotein protein component ofsporozoite membranes that was synthesized by unsporulated oocysts andlater incorporated into sporozoite membranes during the process ofsporulation. Recently, Stotish et al. (73) reported isolation and invitro translation of RNA from unsporulated oocysts, oocysts duringsporulation and from sporozoites. The in vitro translation productsranged from less than 10,000 daltons to greater than 200,000 daltons.Patterns for unsporulated and sporulating oocyst RNA directed-proteinsynthesis were different, suggesting that different RNA populations mayexist during sporulation.

In order to produce cDNA encoding the antigenic proteins, it wasnecessary to determine when the mRNA encoding the antigenic proteinsoccurred during the life cycle of E. tenella. This invention concernsthe isolation and characterization of cDNA clones encoding antigenicproteins and the production of engineered antigenic proteins in E. coli.It also concerns the extraction of these proteins produced in E. colifrom the insoluble state and the process to make the proteinsimmunoreactive with monoclonal antibodies. Finally, this invention showsthe preparation and use of the bacterially produced antigenic proteinsto produce immunity in chickens to coccidiosis caused by E. tenella, E.necatrix and E. maxima.

Antigenic proteins derived from Eimeria tenella and vaccines containingthem for the prevention of coccidiosis caused by E. tenella have beendescribed in European Patent Publication No. 164,176.

SUMMARY OF THE INVENTION

A genomic DNA molecule having the nucleic acid sequence set forth inFIG. 5 and encoding an antigenic protein derived from Eimeria tenellahas been isolated. The native protein has a molecular weight of about25,000 daltons and is composed of two polypeptides joined by a disulfidebond. One of the polypeptides is characterized by a molecular weight ofabout 17,000 daltons and by a blocked N-terminal amino acid and has theamino acid sequence set forth in FIG. 5. The other polypeptide ischaracterized by a molecular weight of about 8,000 daltons and has theamino acid sequence set forth in FIG. 5.

A nucleic acid molecule, which is either cDNA or mRNA, encoding anantigenic polypeptide having a molecular weight of about 25,000 daltonsand having the continuous amino acid sequence set forth in FIG. 7 hasalso been isolated. The cDNA molecule has been inserted into expressionvectors capable of expressing the 25,000 dalton polypeptide directly oras a fused polypeptide.

Vector pDET1 encodes a polypeptide having a molecular weight of about25,000 daltons and the continuous amino acid sequence set forth in FIG.7. This vector was used to transform E. coli host cells and the straindeposited as REN3/pDET1 (ATCC Accession No. 53316).

Vector pDET2 also encodes a polypeptide having a molecular weight ofabout 25,000 daltons and the continuous amino acid sequence set forth inFIG. 7. This vector was used to transform E. coli host cells and thestrain deposited as REN3/pDET2 (ATCC Accession No. 53318).

Vector pBGC23 encodes a fused polypeptide having a molecular weight ofabout 135,000 daltons which has the amino acid sequence of the 25,000dalton polypeptide set forth in FIG. 7 and, at the amino terminal end,the amino acid sequence of beta-galactosidase. This vector was used totransform E. coli host cells and the strain deposited as REN3/pBGC23(ATCC Accession No. 53317).

Vector pCOC12 encodes a fused polypeptide having a molecular weight ofabout 65,600 daltons and having the amino acid sequence of the 25,000dalton polypeptide set forth in FIG. 7 and, at the amino terminal end,the amino acid sequence of prochymosin. This vector was used totransform E. coli host cells and the strain deposited as REN3/pCOC12(ATCC Accession No. 53314).

Vector pCOC20 encodes a fused polypeptide having a molecular weight ofabout 56,500 and having the amino acid sequence of the 25,000 daltonpolypeptide set forth in FIG. 7 and, at the amino terminal end, theamino acid sequence of prochymosin which has an 83 amino acid deletionfrom its natural sequence. This vector was used to transform E. colihost cells and the strain deposited as REN3/pCOC20 (ATCC Accession No.53313).

A method of preparing an antigenic polypeptide, comprises growing any ofthe host cells of the present invention under appropriate conditionspermitting DNA expression and polypeptide production and recovering thepolypeptide so produced under suitable conditions. The recoverycomprises separating the polypeptide from host cells, purifying thepolypeptide, solubilizing the polypeptide, renaturing the polypeptide,and recovering the purified, solubilized, renatured antigenicpolypeptide.

A method of conferring upon a chicken active immunity against infectionby Eimeria tenella comprises administering to a chicken an effectiveimmunizing amount of any of the polypeptides of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 displays the amino acid sequence of the 17,000 dalton polypeptidecomponent of the E. tenella (TA4) antigen determined by microsequencing.FIG. 1 also shows the overlapping peptides produced by various chemicaland enzymatic digestions.

FIG. 2 shows the restriction enzyme map of the E. tenella genomic clone108-1 encoding the TA4 antigen. FIG. 2 also shows the position andorientation of the gene for the TA4 antigen within the 5500 bp E.tenella EcoRI DNA fragment.

FIG. 3 shows the DNA nucleotide sequence of the Bg1 II-EcoRI DNAfragment of the genomic clone 108-1 depicted in FIG. 2. In addition,FIG. 3 shows the amino acid sequence for the signal peptide and the17,000 dalton and the 8,000 dalton polypeptide components of the TA4antigen. FIG. 3 also shows the introns within the gene.

FIG. 4 shows the appearance of the TA4 antigen during sporulation asdetermined by the appearance of a 17,000 dalton subunit immunoreactivewith monoclonal antibody Ptn 9.9 D12.

FIG. 5 shows the DNA nucleotide sequence of the Bg1 II-EcoRI DNAfragment of the E. tenella genomic clone 108-1 encoding the TA4 protein.The amino acid sequence for the signal peptide and the 17,000 and 8,000dalton polypeptide components of the TA4 antigen as it occurs in thesporozoite membrane is also shown. Also shown are the introns within thegene as well as the SacI-PvuII DNA used to identify the mRNA byhybridization, and cDNA clones encoding the TA4 protein.

FIG. 6 shows the occurrence of the TA4 antigen mRNA during sporulation,as determined by hybridization of an internal restriction fragment froma genomic clone of the TA4 gene.

FIG. 7 shows the DNA sequence of the cDNA clone pTCD26 encoding the TA4antigen.

FIG. 8 schematically shows the construction of expression vector pWHA63and the insertion of the DNA from the cDNA clone pTCD26 into expressionvector pWHA63 to generate expression vectors pDET1 and pDET2.

FIG. 9 shows the production of the pDET1/pDET2 protein in Lon⁺ vs. Lon⁻protease deficient strains of E. coli.

FIG. 10 schematically shows the construction of expression vector pBGC23fusing the 3' end of the lac Z gene to the 5' end of the sequenceencoding the cDNA derived antigenic polypeptide.

FIG. 11 shows the production of the pBGC23 protein in E. coli.

FIG. 12 schematically shows the construction of the bovine prochymosinexpression vector pWHA93.

FIG. 13 schematically shows the construction of pCOC12 by fusing the 3'end of the coding sequence of bovine prochymosin to the 5' end of thecoding sequence of the cDNA derived antigenic polypeptide. FIG. 13 alsoshows the derivation of pCOC20 from pCOC12.

FIG. 14 shows the production of the pCOC12 and pCOC20 proteins in E.coli.

FIG. 15 demonstrates the immunoreactivity of the renatured bacterial TA4proteins with monoclonal antibody Ptn 7.2 A4/4.

FIG. 16 displays the restriction enzyme map of the E. necatrix genomicclone 7-49 encoding the NA4 antigen and the position and orientation ofthe gene for the NA4 antigen within the 3900 bp E. necatrix EcoRI DNAfragment.

FIG. 17 shows the DNA nucleotide sequence of 2440 bases of the genomicclone 7-49 depicted in FIG. 16. This sequence includes the entireHindIII-BalI region shown in FIG. 16. Also shown is the amino acidsequence inferred for the E. necatrix NA4 antigen.

FIG. 18 shows the amino acid sequence homology between TA4 and NA4antigens.

FIG. 19 displays the homology of the three introns within the E. tenellaand E. necatrix genes encoding the TA4 and NA4 antigens respectively.

FIG. 20 schmatically shows the construction of the recombinant vectorpSMAC.

FIG. 21 schmetically shows the construction of the recombinant vectorpSS33.

FIG. 22 schmetically shows the construction of the recombinant vectorpNCD.

FIG. 23 schematically shows the construction of expression vectorspTDS1(A) and pTDS2(B).

FIG. 24 schematically shows the construction of expression vectorspDDS1(A) and pDDS2(B).

FIG. 25 shows the relative weight gain in vaccinated vs. nonvaccinatechickens after challenge with E. tenella oocysts.

DETAILED DESCRIPTION OF THE INVENTION

A genomic DNA molecule having the nucleic acid sequence set forth inFIG. 5 and encoding an antigenic protein derived from Eimeria tenellahas been isolated. The native protein has a molecular weight of about25,000 daltons and is composed of two polypeptides joined by a disulfidebond. One of the polypeptides is characterized by a molecular weight ofabout 17,000 daltons and by a blocked N-terminal amino acid and has theamino acid sequence set forth in FIG. 5. The other polypeptide ischaracterized by a molecular weight of about 8,000 daltons and has theamino acid sequence set forth in FIG. 5.

A nucleic acid molecule, which is either cDNA or mRNA, encoding anantigenic polypeptide having a molecular weight of about 25,000 daltonsand having the continuous amino acid sequence set forth in FIG. 7 hasalso been isolated.

It should be understood that "antigenic polypeptide" as the term is usedherein includes preparations prepared under non-reducing conditions asdescribed herein, characterized by the presence within the preparationof a polypeptide having a defined apparent molecular weight on SDS-PAGEunder reducing conditions. When present in such preparations, thepolypeptide may be bound to another component or components, e.g. toanother polypeptide by one or more disulfide bonds or two or moreregions within the polypeptide may be bound to one another, e.g. by adisulfide bond. For those preparations characterized by the presencewithin them of polypeptides with apparent molecular weights of 18,000 orless on SDS-PAGE under reducing conditions the term "fragment" is alsoused to describe such preparations on the assumption that thepreparations include amino acid sequences contained within the intactantigenic protein. In addition the term "fragment" is used to describeamino acid sequences derived from the antigenic protein by proteolyticdigestion.

A DNA molecule encoding an antigenic polypeptide having a molecularweight less than about 25,000 daltons and an amino acid sequenceincluded within the amino acid sequence of the protein encoded by theDNA having the nucleic acid sequence set forth in FIG. 5 iscontemplated. This DNA molecule may also have additional DNA encodinganother amino acid sequence, in which case the molecular weight of thepolypeptide would be increased by the molecular weight of the additionalamino acid sequence.

A DNA molecule encoding an antigenic polypeptide having a molecularweight greater than about 25,000 daltons which comprises the genomic DNAmolecule of the present invention and DNA encoding another amino acidsequence is contemplated.

A DNA molecule encoding an antigenic polypeptide having a molecularweight less than about 25,000 daltons and an amino acid sequenceincluded within the amino acid sequence of the polypeptide encoded bythe DNA having the nucleic acid sequence set forth in FIG. 7 iscontemplated. This DNA molecule may also have additional DNA encodinganother amino acid sequence, in which case its molecular weight would beincreased by the molecular weight of the additional amino acid sequence.

The present invention provides a DNA molecule encoding an antigenicpolypeptide having a molecular weight greater than about 25,000 daltonswhich comprises the nucleic acid molecule set forth in FIG. 7 and DNAencoding another polypeptide amino acid sequence.

A recombinant cloning vehicle comprises cloning vehicle DNA and the cDNAof the present invention. The cloning vehicle DNA being characterized bythe presence of a first and a second restriction enzyme site and thecDNA being cloned into said sites. A cloning vehicle has beenconstructed which contains the cDNA clone, designated pTCD26, of thepresent invention and encodes an antigenic polypeptide having amolecular weight of about 25,000 daltons and the amino acid sequence setforth in FIG. 7. The cloning vehicle may be used to transform abacterial host cell. An E. coli host cell, JM83, has been transformedwith this cloning vehicle and the strain designated as JM83/pTCD26 (ATCCaccession No. 53315).

The present invention contemplates an expression vector capable ofexpressing a 25,000 dalton antigenic protein when introduced into asuitable host cell, which comprises suitable carrier DNA and the genomicDNA set forth in FIG. 5.

When referring to an expression vector carrying the genomic DNA of thepresent invention, a suitable host cell is a euraryotic cell, i.e. ayeast cell or mammalian cell. Otherwise, a suitable host cell is abacterial host cell, i.e. E. coli.

Also contemplated is an expression vector capable of expressing anantigenic polypeptide having a molecular weight less than about 25,000daltons, when introduced into a suitable host cell. The vector comprisessuitable carrier DNA and DNA encoding an antigenic polypeptide having amolecular weight less than about 25,000 daltons and an amino acidsequence included within the amino acid sequence of the protein encodedby the DNA having the nucleic acid sequence set forth in FIG. 5 or FIG.7. The non-carrier DNA may also have additional DNA encoding anotheramino acid sequence, in which case the molecular weight of thepolypeptide would be increased by the molecular weight of the additionalamino acid sequence.

Suitable carrier DNA would be any DNA segment capable of carrying thegenomic DNA molecule of the present invention for use in transformingeucaryotic cells. One such suitable carrier DNA would be that derivedfrom a eucaryotic virus, preferably a commonly used avian virus, such asMarek's disease virus, fowl pox virus or herpes virus of turkeys (HVT)or any mutant derivative thereof.

Also contemplated is an expression vector capable of expressing anantigenic polypeptide having a molecular weight greater than 25,000daltons, when introduced into a suitable host cell, which comprisessuitable carrier DNA and the genomic DNA molecule of the presentinvention and DNA encoding another amino acid sequence.

A bacterial expression vector capable of expressing a 25,000 daltonantigenic polypeptide when introduced into a suitable bacterial hostcell, comprises plasmid DNA and the cDNA of the present invention. Whenunder the control of the lac, lambda P_(R) and tac promoters this vectoris designated pDET1. When under the control of the lac and tac promotersthis vector is designated pDET2.

A suitable bacterial expression vector is a double-stranded DNA moleculewhich includes in 5' to 3' order the following:

a DNA sequence which contains a promoter and operator or just apromoter;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the desired gene capable of binding to ribosomes within the hostcell

an ATG initiation codon;

a restriction enzyme site for inserting a desired gene into the vectorin phase with the ATG initiation codon;

a DNA sequence which contains an origin of replication from a bacterialplasmid capable of autonomous replication in the host cell; and

a DNA sequence which contains a gene associated with a selectable oridentifiable phenotypic trait and which is manifested when the vector ispresent in the host cell.

The present invention contemplates a bacterial expression vector capableof expressing an antigenic polypeptide having a molecular weight lessthan about 25,000 daltons when introduced into a suitable bacterial hostcell. The vector comprises plasmid DNA and DNA which encodes apolypeptide having an amino acid sequence included within the amino acidsequence set forth in FIG. 7. The non-plasmid DNA may also haveadditional DNA encoding another amino acid sequence, in which case themolecular weight of the polypeptide would be increased by the molecularweight of the additional amino acid sequence.

The present invention provides a bacterial expression vector capable ofexpressing a fused polypeptide composed a polypeptide of about 25,000daltons fused to another amino acid sequence when introduced into asuitable host cell. It comprises plasmid DNA and the cDNA of the presentinvention fused to DNA encoding another amino acid sequence.

The vector pBGC23 encodes an antigenic fused polypeptide having amolecular weight of about 135,000 daltons and having the amino acidsequence of beta-galactosidase fused to the amino terminal end of theamino acid sequence set forth in FIG. 7.

The vector pCOC12 encodes an antigenic fused polypeptide having amolecular weight of about 65,600 daltons and having the amino acidsequence of prochymosin fused to the amino terminal end of the aminoacid sequence set forth in FIG. 7.

The vector pCOC20 encodes an antigenic fused polypeptide having amolecular weight of about 56,500 daltons and having the amino acidsequence of prochymosin, which has an 83 amino acid deletion from itsnatural sequence, fused to the amino terminal sequence set forth in FIG.7.

The bacterial expression vectors of the present invention have been usedto transform E. coli host cells. The E. coli host cell designatedREN3/pBGC23 comprises the vector pBGC23 and has ATCC accession No.53317. The E. coli host cell designated REN3/pCOC12 comprises the vectorpCOC12 and has ATCC accession No. 53314. The E. coli host celldesignated REN3/pCOC20 comprises the vector pCOC20 and has ATCCaccession No. 53313. The E. coli host cell designated REN3/pDET1comprises the vector pDET1 and has ATCC accession No. 53316. The E. colihost cell designated REN3/pDET2 comprises the vector pDET2 and has ATCCaccession No. 53318.

A method of preparing an antigenic polypeptide, comprises growing any ofthe host cells of the present invention under appropriate conditionspermitting DNA expression and polypeptide production and recovering thepolypeptide so produced under suitable conditions. The recovery stepcomprises first separating the polypeptide from host cells and thenpurifying it, solubilizing it, renaturing it and finally recovering thepurified, solubilized, renatured antigenic polypeptide.

A method of conferring upon a chicken active immunity against infectionby Eimeria tenella comprises administering to a chicken an effectiveimmunizing amount of any of the polypeptides of the present invention.The polypeptides may also be administered in any combination of two ormore polypeptides.

A vaccine for conferring upon a chicken active immunity againstinfection by Eimeria tenella comprises per dose an effective immunizingamount of any one of the polypeptides of the present invention and asuitable carrier. The vaccine may also comprise a combination of two ormore polypeptides of the present invention and a suitable carrier. Inone embodiment, the polypeptide used in the vaccine is the fusedpolypeptide having a molecular weight of about 135,000 daltons and theamino acid sequence of beta-galactosidase fused to the amino terminalend of the amino acid set forth in FIG. 7. In another embodiment thepolypeptide used in the vaccine is the fused polypeptide having amolecular weight of about 65,600 daltons and having the amino acidsequence of prochymosin fused to the amino terminal end of the aminoacid sequence set forth in FIG. 7.

A method of protecting a chicken against infection by Eimeria tenellacomprises administering to the chicken a suitable dose of any of thevaccines of the present invention.

Plasmid pDET1 encodes a 25,000 dalton polypeptide under the control ofthe lac, lambda P_(R) and tac promoters. Plasmid pDET2 encodes a 25,000dalton polypeptide under the control of the lac and tac promoters (FIG.8). The greatest yield of the pDET1/pDET2 proteins was achieved in aprotease deficient E. coli strain (FIG. 9). The pDET1 and pDET2 proteinswere found in the insoluble fraction of a cell lysate.

Plasmid pBGC23 was constructed by fusing the 3' end of the codingsequence of E. coli beta-galactosidase to the 5' end of the codingsequence of the cDNA derived TA4 polypeptide and encodes a fusionprotein of approximately 135,000 daltons (FIG. 10). The pBGC23 proteinis stable but insoluble in E. coli (FIG. 11).

Plasmid pCOC12 was constructed by fusing the 3' end of the codingsequence of bovine prochymosin to the 5' end of the cDNA coding for theTA4 polypeptide and encodes a fusion protein of approximately 65,600daltons. Plasmid pCOC20 was constructed from pCOC12 by a deletion in theprochymosin domain of the fusion protein and encodes a fusion protein ofapproximately 56,500 daltons (FIG. 13). The pCOC12 and pCOC20 proteinsare stable but insoluble in E. coli (FIG. 14).

The insoluble, bacterially-produced TA4 proteins were not immunoreactivewith Ptn 7.2 A4/4, a neutralizing monoclonal raised to E. tenellasporozoites. When the insoluble proteins from pBGC23 and pCOC12 wereinjected into mice they did not raise antibodies that crossreacted withthe TA4 antigen purified from E. tenella.

This invention also concerns a method for extracting thebacterially-produced TA4 proteins from the insoluble state and theprocess to make the proteins immunoreactive with monoclonal antibody Ptn7.2 A4/4. This method is applicable to renaturation of prochymosin-TA4fusion proteins to make them immunoreactive. It involves solubilizationof the proteins in 8M urea followed by dilution and renaturation atalkaline pH (pH 11) and back titration to pH 8.3. Alternatively theproteins may be solubilized in 8M urea and the urea removed by dialysis.

When the urea-alkali solubilization/renaturation process was used forthe pCOC12 protein the renatured protein had both milk clotting activityand immunoreactivity with monoclonal antibody Ptn 7.2 A4/4. Renaturationconditions were optimized using the pCOC12 protein. The optimalrenaturation conditions for pCOC20 protein and pBGC23 protein were foundto be the same as those for pCOC12. For pDET2 protein on the other handoptimal renaturation conditions involved urea-dialysis at alkaline pH.

The renatured pBGC23 and pCOC12 proteins elicited antibodies in micethat reacted with the TA4 antigen purified from E. tenella. Whenchickens were immunized with renatured pBGC23 and pCOC12 proteins theseproteins elicited serum neutralizing antibodies to E. tenellasporozoites and ameliorated coccidiosis in chickens challenged with E.tenella.

This invention also encompasses a method for conferring upon a chickenactive immunity against infection by Eimeria tenella which comprisesadministering to a chicken an effective immunizing amount of therenatured bacterial TA4 proteins. By this method active immunity can beconferred upon a non-immune chicken. In addition, administration ofthese materials can be used to increase a relatively low level ofimmunity in a chicken previously exposed to E. tenella and can be usedin booster vaccinations.

The bacterial TA4 proteins can be administered to chickens by any of anumber of well known methods. Desirably, the administration can involvesubcutaneous, intraperitoneal or intramuscular injection at the back ofthe neck, or any convenient form of oral administration. The amount ofantigen comprising an effective immunizing amount can be any amount fromabout 0.1 microgram to about 1 mg. The amount of antigen is desirablyabove about 10 micrograms. The preferred amount of antigen is about 500micrograms per kilogram of body weight. Alternatively, theadministration can be oral (e.g., via capsule) or desirably by injection(e.g., subcutaneous, intradermal, or preferably intramuscularinjections). If the mode of administration involves injection, anypharmaceutically acceptable carrier can be employed. Suitable carriersinclude 0.01 to 0.1M, preferably 0.05M, phosphate buffer or 0.8 percentsaline.

A vaccine for conferring upon a chicken active immunity againstinfection by Eimeria tenella is provided which comprises an effectiveimmunizing amount of an antigenic material of this invention, i.e., therenatured bacterial TA4 proteins and a suitable carrier. Preferably theeffective immunizing amount of the antigenic material in the vaccine isabove about 0.1 microgram/kg of body weight of the chicken.

In addition, the carrier desirably also contains a preservative. Oneparticularly suitable preservative is thimerosal (sodiumethylmercurithiosalicylate) which has activity as both a bacteriostatand a fungistat. Desirably, thimerosal is present in the vaccine in afinal concentration of 10⁻⁴ percent.

Furthermore, the carrier desirably also contains an immunopotentiator,i.e., a substance which enhances the immune response of the treatedanimal to the vaccine, including Salmonella minnesota LPS at 10micrograms/dose. Various immunopotentiators known in the art may beused. The adjuvant presently employed is 94% Drakeol 6-VR, 5% Arlacel A,1% Tween-80. Arlacel A is a mannide monoleate (Sandria Corp.). It is anirritant which has strong immunopotentiating activity when combined withantigens. Drakeol 6-VR is a hypoallergenic light mineral oil product(Penreco Corp.). Tween-80 is a monoleate derivative ofpolyoxyethylsorbitan and possesses detergent properties. Other suitablecarriers or immunopotentiators include aluminum potassium sulfate,aluminum hydroxide, ligand binding subunits of toxin molecules,bioadhesives, lymphokines and water in oil emulsions.

By administering a suitable dose of such a vaccine to a chicken, thechicken is protected against infection by E. tenella. The amount ofantigenic material per dose should be sufficient to induce production ofantibodies to the antigenic material in an animal to which the vaccineis administered. To provide a sufficient degree of immunologicalresponse as measured by antibody production and protection, the amountof the antigenic material per dose is desirably above about 20.0micrograms/kg of body weight of the vaccinated animal. Thus, the amountof antigenic material based upon a 50 gram day-old chick would be aboveabout 1.0 microgram. Presently preferred is a vaccine containing 10micrograms of antigenic material. In general, the antigen will compriseon a weight basis from about 0.002 percent up to about 0.2 percent ofthe vaccine and the dose volume will be about 0.1 ml.

EXAMPLE 1 Preparation of Eimeria necatrix and Eimeria tenella Oocysts,Sporocysts and Sporozoites

Coccidia. The purified field isolates of Eimeria necatrix and Eimeriatenella were originally purchased from Dr. Allen Edgar of the Universityof Auburn. The purity of each isolate was confirmed using oocystcharacteristics and histology of infected intestinal tissue. Oocyst sizeand shape index were within the range of E. necatrix and E. tenella,respectively.

Lesions were scored by the method of Johnson and Reid (30) The lesionsin infected birds were typical of each respective isolate. At 5 dayspost-infection histological examination revealed larger secondgeneration schizonts in the subepithelium of the mid-intestine (E.necatrix) or the ceca (E. tenella). Mortality was experienced with E.tenella and E. necatrix during severe infections (15,000 and 50,000oocysts respectively). Single oocyst cloning was periodically done toinsure purity of each isolate.

Propagation of Oocysts. Pure cultures of each isolate were routinelypassaged in 4- to 6-week old SPF white Leghorn chickens. To avoidextraneous coccidial infections, chickens were reared from 1 day of agein plexiglass isolation units. Oocysts were harvested on day 7post-infection from the ceca using a trypsin-digest method described byShirley (66). Sporulated oocysts were typically stored at 24° C. in 2%w/v K₂ Cr₂ O₇.

Isolation of Sporocysts. Sporulated oocysts, (1×10⁸) which had beenpartially purified from debris by salt floatation, were washed fivetimes in 0.1M phosphate buffered saline, pH 7.4, (PBS) to remove thepotassium dichromate preservative. These oocysts were further cleaned byagitation in a 1.05% sodium hypochlorite solution for 20 minutesfollowed by five washes in PBS to remove residual sodium hypochloriteand debris. Following the final wash, the cleaned oocysts wereresuspended in 10 ml of PBS. Suspended oocysts were then mechanicallybroken by shaking with an equal volume of glass beads (1.0-1.05 mm). Theliberated sporocysts were purified from the oocyst walls and fromunbroken oocysts by passage over a glass wool column, centrifuged at3,000 RPM for ten minutes at 4° C. and resuspended in 10 ml of PBS.

Preparation of Sporozoites. Freshly sporulated oocysts were cleaned bysalt floatation, repeated washing and treatment with 1.05% sodiumhypochlorite solution. Sporocysts were freed by mechanically breakingoocysts with glass beads (1.0-1.05 mm). To excyst sporozoites,sporocysts were incubated with trypsin and taurodeoxycholic acid (0.25and 0.50% w/v, respectively) for a period of 1 hour at 41° C.Sporozoites thus obtained were rinsed free of excysting fluid bycentrifugation and resuspended in Hank's medium. Fresh Hank's medium wasused to dilute sporozoites to the working concentration.

EXAMPLE 2 Generation, Identification and Characterization of Hybridomas

Monoclonal Antibody. Monoclonal antibodies were derived from hybridomasdeveloped using the method of VanDeusen and Whetstone (77). Briefly,Balb/C ByJ mice were repeatedly immunized with 10⁶ -10₇ intact E.tenella sporozoites. Three days after a final intravenous injection withintact sporozoites, a randomly selected mouse was sacrificed andsplenectomized. The splenocytes were separated from fibrous tissue inthe organ, and the washed cells fused with the murine plasmacytoma cellline (SP2/OM).

Microneutralization Assay. The microneutralization assay was performedwith primary chick kidney cell cultures for E. tenella or embryonicporcine lung cells for E. necatrix. 1- to 2-week-old chicks weresacrificed and aseptically nephrectomized. The cells were plated into96-well cultures at a density of approximately 10⁴ /well in Earle's LAHmedium supplemented with 5% heat-inactivated fetal calf serum. Cultureswere maintained at 41° C. in a 5% CO₂ atmosphere. When cell culturesreached a level of approximately 50% confluency, 50 microliters ofhybridoma test or control sample were added to all wells of the plate.Next, about 3×10⁴ sporozoites suspended in 50 microliters of Earle'sculture medium were added to all wells of the plate. Twelve to sixteenhours later, the culture supernatant was replaced with fresh Earle's LAHcontaining 2% heat inactivated fetal calf serum. The cultures wereterminated at 40-44 hours post-infection. Culture supernatant wasemptied from the plates at that time. Subsequently, cells were fixed tothe plates by the addition of methanol acidified with 5% glacial aceticacid. The fixed cultures were stained with 0.1% toluidine blue beforeexamination. Wells were scored as to the approximate percentage level ofinhibition of schizogony; neutralization of parasites by monoclonalantibodies was scored on the basis of the maximum serum dilution stillaffording complete inhibition of schizont development.

Indirect Fluorescent Antibody Screening. IFA slides were prepared withsporozoites of E. tenella or E. necatrix (about 1×10⁶ /well). Slideswere air dried several hours to overnight before 10 microliters of 1%bovine serum albumin (BSA) was added to each well. Five minutes afteradding BSA, 20 microliters of test supernatant was added. Supernatantswere incubated at 37° C. for 20 minutes, followed by three rinses with0.15M PBS with 0.0005% Tween-20 (PBS-Tween). Fluorescein conjugatedrabbit anti-mouse antibody (diluted 1:40 in PBS) was added to thesamples and allowed to incubate at 37° C. for 20 minutes. The conjugatewas rinsed off three times with PBS-Tween before adding mounting mediumand cover slip.

Results. Of the several thousand hybridomas developed against Eimeriatenella, 24 were found to produce neutralizing antibodies toward thesporozoite stage of the parasite. All of the hybridomas studied producedantibodies that recognized membrane bound antigens, although only theantibody produced by one hybridoma recognized an internal membraneantigen.

In vitro neutralizing potency was compared for several supernatantsafter the initial cloning of the respective cell lines. Supernatant fromcertain lines demonstrated the greatest relative propensity forneutralizing sporozoites of E. tenella. When antibody content wasassessed for each of the supernatants tested, it was determined thattwenty-fold less of one antibody (designated Ptn 7.2A4/4) was requiredto neutralize sporozoites than the second most effective neutralizingantibody. Specifically, the amount of Ptn 7.2A4/4 antibody required toneutralize E. tenella is approximately 3.5×10⁵ molecules/sporozoite.

The hybridoma which produces the monoclonal antibody designated Ptn7.2A4/4 has been deposited with the American Type Culture Collection inRockville, Md., U.S.A. 20852, and identified by ATCC accession No.HB8561. This deposit was made pursuant to the provisions of the BudapestTreaty on the International Recognition of the Deposit of MicroorganismsFor The Purposes Of Patent Procedure (hereinafter "Budapest Treaty").

When the monoclonal antibody Ptn 7.2A4/4 was evaluated with E. necatrix,it was observed that a fluorescent staining pattern, similar to thatwith E. tenella had developed. The monoclonal was therefore studied inthe in vitro neutralization assay against E. necatrix. Said monoclonalantibody was found to possess neutralizing activity against E. necatrixat levels within a comparable range observed with a like number of E.tenella sporozoites.

EXAMPLE 3 Identification of the Antigens of E. Tenella Recognized byNeutralizing Monoclonal Antibody Ptn 7.2A4/4

¹²⁵ I Labeling of Eimeria Proteins. A total of 2×10⁸ oocysts from E.tenella were processed for iodination. In each case, sporocysts werepurified from salt floated, sodium hypochlorite treated oocysts thatwere broken with glass beads then passed through a glass wool column.Sporocyst membranes were prepared from one-half of the sporocysts bymechanical breakage in 1 ml 10 mM sodium phosphate, 0.15M NaCl, pH 7.2(PBS) with glass beads in the presence of protease inhibitors: 0.1 mMPhenylmethlysulfonyl fluoride (PMSF), 0.1 mM N-tosyl-L-phenylalaninechloromethyl ketone (TPCK), 1 mM N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK) and 10 KIU/ml aprotinin. The remaining sporocysts weretreated with trypsin and taurodeoxycholic acid (total volume=1 ml) toexcyst sporozoites. Both preparations were pelleted at 45,000 RPM for 45minutes at 4° C. and resuspended in 1 ml of phosphate buffered saline(PBS). Care was taken to remove all trypsin - deoxycholate residue fromthe sporozoites by washing with PBS and 1 mM PMSF prior toultra-centifugation.

The one ml samples were put into glass scintillation vials which hadbeen coated with 40 micrograms of IODOGEN(1,3,4,6-tetrachloro-3-alpha,6-alpha-diphenylglycouril) solid phaseiodination reagent (24, 53), dried under nitrogen gas and rinsed withPBS. To each tube, 0.5 mCi of ¹²⁵ I was added and the samples allowed toincubate for 20 minutes on ice. Afterward, 100 microliters of KI (1M)was added to each tube to a final concentration of 100 mM, and thereaction was allowed to proceed for an additional 15 minutes on ice.Sporozoite and sporocyst preparations were then diluted to 7 ml with PBScontaining 5 mM KI and pelleted at 45,000 RPM for 45 minutes at 4° C.

Extraction of Sporocyst and Sporozoite Membrane Proteins. ¹²⁵ I labeledsporocyst and sporozoite pellets from the above high speedcentrifugation were resuspended in 1 ml of protein extraction buffer (20mM Tris-HCl, pH 7.5; 50 mM MgCl₂ ; 25 mM NaCl, 1% NP40, 1 mM PMSF, 0.1mM TPCK, 1 mM TLCK and 10 KIU/ml aprotinin). The suspensions wereincubated for 60 minutes on ice with occasional vortexing. Insolublematerial was separated from the detergent solubilized protein in amicrofuge for 15 minutes at 4° C. The supernatants were stored at -70°C.

TCA Precipitation of ¹²⁵ I Proteins. Ten microliters of each sample werediluted into 90 microliters of 5 mM KI. Ten microliters of each dilutedsample was then added to a solution containing 1 ml of 5%trichloroacetic acid (TCA), 25 microliters BSA (10 mg/ml) and 5 mM KIand incubated on ice for 30 minutes. The precipitated samples werecollected by filtration through glass fiber filters, washed twice with 5ml of 5% TCA, 5 mM KI and three times with 5 ml of 95% ethanol, both at0° C., and counted in a liquid scintillation counter.

Immunoprecipitation With Monoclonal Antibodies: Fifty microliters ofmonoclonal antibody were added to 25 microliters of monoclonal antibodydilution buffer (MAB-DIL): 50 mM Tris-HCl, pH 8.6; 150 mM NaCl; 0.1%NP-40; 0.1% BSA, RIA grade; 1 mM TLCK; 1 mM PMSF; 10 KIU/ml aprotinin.Twenty microliters of ¹²⁵ I labeled protein was then added and the tubevortexed and incubated overnight at 4° C. Rabbit anti-mouse Ig serum(IgA, IgG, IgM) was diluted 1:2 in MAB-DIL and 10 microliters added toeach immunoprecipitation tube and incubated 1 hour at 4° C. ProteinA-Sepharose (10% v/v) was diluted 1:4 in monoclonal antibody washbuffer, (MABW): 50 mM Tris-HCl, pH 8.3; 0.05% NP-40; 0.05% Triton X-100;150 mM NaCl; 0.02% NaN₃ ; 5 mM KI and 400 microliters added to eachtube. The tubes were incubated for one hour at 4° C. with gentlerocking. The immunoprecipitation products were washed twice with coldMABW followed by two room temperature washes with MABW. The pellet wasresuspended in 50 microliters of SDS-PAGE sample buffer (35), boiled for5 minutes and microfuged to remove the protein A-Sepharose. Supernatantswere counted and analyzed by SDS-PAGE.

Electrophoretic Transfer of Antigens to Nitrocellulose Paper:Uniodinated sporozoite membrane proteins (detergent solubilized asalready described) were separated under either reducing or nonreducingconditions by one dimensional sodium dodecyl sulfate polyacrylamide slabgels and electrophoretically transferred to nitrocellulose paper (75).Electrophoretic blots were processed according to the method of Sharmaet al (64) with the exceptions that sera, monoclonal antibodies and theappropriate conjugates (peroxidase conjugated goat anti-chicken IgG,Kirkegaard and Perry, peroxidase conjugated rabbit anti-mouse IgG(Cappel) were employed for blots of reducing gels, and murine monoclonalantibodies used in conjunction with the Vectastain ABC kit for mouse IgGfor nonreducing gels (Vector Labs, Burlington, Calif). Blots weredeveloped by reacting them with 4-chloro-1-napthol (Sigma; 660micrograms/ml) and H₂ O₂ (0 17%) for reduced separation or Vectastainreagents for nonreducing separations.

SDS - Polyacrylamide Gel Electrophoresis (SDS-PAGE) of E. tenellaProteins. Total ¹²⁵ I labeled sporocyst and sporozoite membrane proteinsimmunosorbed, and immunoprecipitated proteins were analyzed on, 5-25%exponential or 8-20% linear gradients SDS-polyacrylamide gels at 25 mA.The gels were dried and exposed to Kodak XAR-5 X-ray film overnight at-70° C. Gels used for staining purposes were visualized by Coomassie(21) or silver staining using the manufacturer's labelled instructions(Pierce Chemical).

Results of Immunoprecipitation of E. tenella Antigen with Ptn 7.2A4/4Monoclonal Antibody. The surface-labeled E. tenella sporozoitepreparation contains two heavily iodinated proteins with apparentmolecular weights of 6,500 and 25,000 as judged on reducing SDS-PAGE.The 6,500 dalton protein is readily and specifically immunoprecipitatedwith monoclonal antibody Ptn 7.2A4/4. Membranes from sporocysts containtwo heavily iodinated proteins with apparent molecular weights of 17,000and 27,000 although several other minor iodinated proteins of variousmolecular weights are also present. Upon immunoprecipitation of ¹²⁵ Ilabeled sporocyst membrane protein the only antigen precipitatedfollowing the reaction with the monoclonal antibody Ptn 7.2A4/4 was the17,000 dalton protein as determined on reducing SDS-PAGE.

Results of Western Blots of E. tenella Antigens with Ptn 7.2A4/4Monoclonal Antibody. Under the conditions in which theimmunoprecipitated, iodinated polypeptides were analyzed on SDS-PAGE asdescribed above, polypeptides linked by disulfide bonds have beenseparated. However, reduction of disulfide bonds destroys Ptn 7.2A4/4reactivity on Western blots in both sporocyst and sporozoite membranepreparations. When iodinated sporocyst and sporozoite membranepreparations were run on SDS-PAGE under non-reducing conditions themajor radiolabeled species migrates with an apparent molecular weight of23-25,000. Furthermore, this apparent 23-25,000 dalton species wasreactive with monoclonal antibody Ptn 7.2A4/4 by Western blotting. Theseresults suggest that the 17,000 dalton polypeptide and the 8,000 daltonpolypeptide are complexed together to form the TA4 antigen. The factthat this other polypeptide component of the TA4 antigen was notobserved in immunoprecipitation experiments of iodinated material can beexplained by the observation that this other polypeptide does notcontain any tyrosines that could be iodinated (see description of the8,000 dalton polypeptide component of the TA4 antigen in Examples 5 and6).

EXAMPLE 4 Purification, Identification and Characterization of the E.Tenella TA4 Antigen and Fragments Containing Fractions Thereof

Purification of the 17,000 Dalton Peptide Component of the TA4 Antigen.E. tenella sporulated oocysts were resuspended in 10 ml PBS per 10⁹oocysts and were broken by shaking with an equal volume of glass beads.Membranes were isolated by centrifugation (100,000×g, 60 min., 4° C.)and the proteins were solubilized in 1% (v/v) NP-40, 10 mM Tris-HCl (pH7.5), 25 mM NaCl, 1 mM PMSF, 1 mM TLCK, 0.1 mM TPCK and 10 KIU/mlaprotinin. Insoluble material was pelleted with another 100,000×g spin(60 min., 4° C.). The protein was adsorbed to a DEAE-cellulose columnequilibrated with 10 mM Tris-HCl (pH 7.7), 0.05% NP-40 and then washedwith this buffer containing 50 mM NaCl. After elution with buffercontaining 200 mM NaCl, the 17,000 dalton polypeptide was concentratedby acetone precipitation and the precipitate resuspended in loadingbuffer, boiled and subjected to electrophoresis in SDS-polyacrylamide(15%). Conventional SDS-PAGE sample buffer used in this and otherexperiments contained 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) sodium dodecylsulfate, 10% (w/v) glycerol and 0.001% (w/v) bromphenol blue. The bufferalso contained 5% (v/v) beta-mercaptoethanol except in experiments inwhich non-reducing conditions are specified. The 17,000 daltonpolypeptide band was identified by staining (Coomassie blue or KCl). Theappropriate gel region was excised, the protein electroeluted andconcentrated by acetone precipitation. Note that these procedures aredenaturing for proteins and peptides bound to each other by disulfidebonds are separated with this method. The 17,000 dalton polypeptidepurified by this method was essentially pure.

Purification and Characterization of the TA4 Antigen. As an alternativeto purification by gel electrophoresis the sporocyst membrane proteinsfrom the DEAE-cellulose column were dialyzed against 10 mM Tris-HCl,pH8, 0.05% NP-40 and applied to a DEAE-HPLC column (Bio-Rad)equilibrated in this buffer. The column was eluted with a NaCl gradient(0-300 mM) in the same buffer. The 17,000 dalton polypeptide (identifiedby its migration on gel electrophoresis) was found in material elutingat 200 mM NaCl. Fractions containing this protein were applied to ahydroxyapatite column (HPHT-Bio-Rad) equilibrated with 30 mM potassiumphosphate, pH 6.5, 0.05% Zwittergent 3-12 (Calbiochem-Behring, LaJolla,Calif.), 0.1 mM dithiothreitol. The column was washed with equilibrationbuffer and developed with a potassium phosphate gradient (0-300 mM)containing 0.05% Zwittergent and 0.1 mM dithiothreitol. The 7,000 daltonpolypeptide (identified by gel electrophoresis described above) appearedin material eluting at approximately 90 mM potassium phosphate.

Fractions containing the 17,000 dalton polypeptide purified by thismethod also contained a second peptide of 8,000 daltons. This peptideappears to be linked by a disulfide bridge to the 17,000 daltonpolypeptide. If the fractions containing the 17,000 dalton peptide wereimmunoprecipitated with monoclonal antibody Ptn 7.2A4/4 and theprecipitated proteins analyzed by gel electrophoresis under reducingconditions (as above) both the 17,000 and 8,000 dalton polypeptidesappear to be immunoprecipitated. Hence, in sporocyst membranepreparations, the 8,000 dalton and 17,000 dalton polypeptides appear tobe linked by a disulfide bond (presumably by a cysteine bridge) becausethe two peptides did not appear on electrophoresis unless a strongreducing agent was present. Under nonreducing conditions, the Ptn7.2A4/4 reactive species migrates with an apparent molecular weight of21-24,000.

Preparation of the 11,500 dalton fragment of the TA4 antigen. E. tenellasporocyst membranes were prepared as described above and resuspended in10 ml of PBS+1% Triton X-100. To this 10 ml membrane suspension wasadded 10 ml of 80% phenol containing 0.1% 8-hydroxyquinoline. Thesuspension was then vortexed at maximum at 4000 RPM. The phenol and theflocculent interface were removed and diluted in five volumes of 100 mMammonium acetate in methanol and allowed to precipitate at -20° C.overnight. Following two washes in acetone, the insoluble proteins wereagitated for 8 hours in 0.5% SDS, and insoluble materials removed bycentrifugation at 20,000 RPM for one hour at 4° C. The sample wasdialyzed extensively against PBS (pH 7.2) containing AG 501-X8 mixed bedresin (1 gm/500 ml). The 11,500 dalton fragment of the TA4 antigen wasthen immunoadsorbed from the supernatant using the Ptn 7.2A4/4monoclonal antibody as follows. This polypeptide was shown to bereactive with the Ptn 7.2A4/4 monoclonal antibody by microtiter plateELISA.

For microtiter plate ELISA polystyrene 96 well clusters (Immulon II)were sensitized with antigen in 10 mM glycine buffered saline, pH 9.6,incubated overnight at 37° C. The wells were washed with 0.15M PBS with0.0005% Tween-20, blocked with 3% BSA in PBS-Tween, rewashed, andincubated with Ptn 7.2A4/4 monoclonal antibody diluted in PBS. The wellswere washed as before, and then incubated with peroxidase conjugatedrabbit anti-mouse IgG serum diluted in PBS. The wells were washed againand then incubated with substrate (2,2'-azino-di-[3-ethyl-benzthiazolinesulfonate]) in the presence of H₂ O_(O) ₂. Color development wasdetermined with a Dynatech MR-580 microtiter plate ELISA reader after 15minutes. The 11,500 dalton fragment of the TA4 antigen was shown to bereactive with the Ptn 7.2A4/4 monoclonal antibody by microtiter plateELISA.

EXAMPLE 5 Amino Acid Sequence of the 17,000 and 8,000 Dalton PeptideComponents of the E. Tenella TA4 Antigen

Amino Acid Sequence of the 17,000 Dalton Peptide Component of the TA4Antigen. Amino acid sequencing of the 17,000 dalton peptide wascomplicated by the finding that the N-terminal amino acid was blocked(i.e. not accessible to Edman degradation (14)). To circumvent thisproblem the protein was reduced and alkylated and then digested withvarious chemicals and enzymes. The resulting peptides were purified byreverse phase HPLC (26). The 17,000 dalton polypeptide or the TA4antigen was digested with CNBr (CN), V8 protease (V), chymotrypsin (CH)and Endoprotease Arg-C (R).

Before protease digestion the purified 18,000 dalton polypeptide or theTA4 antigen was treated with 30 mM dithiothreitol, 6M guanidine-HCl (pH8) for 1 hour at room temperature. Solid iodoacetamide was added to afinal concentration of 100 mM, the pH was readjusted to 8 and the samplewas incubated for 1 hour at room temperature. Following reduction andalkylation, samples were purified from reagents either by P6DG (Bio-Rad,Richmond, Calif.) spin columns equilibrated in 0.1M MOPS, pH 7.5, 0.1%SDS or by reverse phase HPLC.

For CNBr digestion, the protein sample was treated with 1% CNBr in 70%formic acid for 20 hours at 4° C. The sample was evaporated to drynessin a Savant Speedvac centrifuge and redissolved in 0.1% trifluoroaceticacid (TFA) or 0.1% TFA, 20% acetonitrile (CH₃ CN). V8 digestion wasperformed in 0.1% SDS, 0.1M MOPS pH 7.5 for 2 hours at room temperatureat a ratio of 50 micrograms 17,000 dalton polypeptide: 1 microgram V8.After digestion, the samples were precipitated with 4 volumes of acetoneat -20° C. overnight. The acetone precipitates were redissolved asdescribed above. Chymotrypsin digestion was performed in 0.05%Zwittergent 3-12, 0.1M NH₄ HCO₃, pH 7.8 for 1 hour at 37° C. at a ratioof 50:1, 17,000 dalton peptide:chymotrypsin. Samples were acidified withTFA for peptide purification. Arg-C digestion was performed in 0.05%Zwittergent 3-12, 0.1M NH pH 7.8 for 2 hours at 37° C. at a ratio of15:1, 17,000 dalton peptide: Arg-C. After acetone precipitationovernight at -20° C., the peptides were mainly in the acetonesupernatant. The supernatant was evaporated and the samples redissolvedas described above. Peptides were purified on a Vydac C4 column (theSeparations Groups, Inc., Hesperia, Calif.) and eluted with a 0-100% CHCN gradient in 0.1% TFA.

Amino acid sequencing was performed using a gas phase sequencer (AppliedBiosystems, Inc., Foster City, Calif.) according to the procedure ofHunkapiller et al (25). Phenylthiohydantoin (PTH) derivatized aminoacids were analyzed by HPLC (8).

The N-terminal amino acid was determined directly by removing theblocking agent. The 17,000 dalton peptide was treated with pyroglutamateaminopeptidase (5:1 protein:PAP) in 0.1M potassium phosphate (pH 8.0),10 mM EDTA, 5% glycerol, 5 mM dithiothreitol, 0.05% Zwittergent® 3-12for 1 hour at 37° C. After treatment, the amino acid sequence could bedetermined directly suggesting that the N-terminal amino acid glutamineis cyclized to form the blocked residue pyrrolidone carboxylic acid. Thecomplete amino acid sequence for the 17,000 dalton peptide component ofthe TA4 antigen is shown in FIG. 1.

Partial Amino Acid Sequence of the 8,000 Dalton Peptide Component of theTA4 Antigen. When the purified 8,000 dalton peptide (derived from theTA4 antigen by reduction and alkylation) was subjected to Edmansequencing the N-terminal amino acid sequence could be determineddirectly. A partial amino acid sequence of the N-terminal region of thepeptide is shown below. ##STR1##

EXAMPLE 6 Isolation and Characterization of a Genomic DNA Clone Encodingthe Eimeria Tenella TA4 Antigen

Isolation of DNA from E. tenella Sporulated Oocysts. Sporulated oocysts(5×10⁸) were washed and sporocysts were isolated as describedpreviously. Isolated sporocysts were washed 2× with 0.1M Tris-HCL, (pH8.5), 0.2M NaCl, 10 mM EDTA. Sporocysts were lysed by incubation for 30min. at 65° C. in 0.1M Tris-HCl, (pH 8.5), 0.2M NaCl, 50 mM EDTA, 1%SDS, 150 micrograms/ml Proteinase K. After cooling to room temperaturethe DNA was gently extracted with an equal volume of liquified phenolfor 1 hour. After centrifugation for 10 min. at 3,000 rpm, the aqueouslayer was removed and the interface and phenol were re-extracted with 10mM Tris-HCl (pH 8), 1 mM EDTA. The aqueous phases were pooled andextracted 1× with phenol and 2× with chloroform:isoamyl alcohol (24:1).DNA was isolated by ethanol precipitation. The DNA pellet wasredissolved in 10 mM Tris-HCl (pH 8), 1 mM EDTA and treated with 0.15mg/ml DNase free-RNase A for 1 hour at 37° C. After RNase digestion, thesample was extracted 1× with phenol, 1× with chloroform: isoamyl alcoholand then precipitated with ethanol. On agarose gels, the size of the DNAwas determined to be greater than 20 kilobase pairs.

Construction of the E. tenella Genomic Library in Bacteriophage λgt wesλB. The E. tenella genomic DNA library in bacteriophage λgt wes λB (36)was constructed using methods described by Maniatis et al. (44). Phagewere purified by polyethyleneglycol precipitation, chloroform extractionand CsCl gradient centrifugation. Purified phage were disrupted with 1%SDS, 50 mM EDTA and 150 micrograms/ml Proteinase K, and DNA was purifiedby phenol extraction, chloroform extraction and ethanol precipitation.The E. tenella genomic DNA and phage DNA were digested to completionwith EcoRI. The left and right arms of the phage DNA were annealed attheir cohesive ends and the arms were purified by sucrose densitygradient centrifugation. 30 micrograms of EcoRI digested DNA arms wereligated to 6 micrograms of EcoRI digested E. tenella DNA using T4 DNAligase. 20 micrograms of the ligated DNA were packaged in vitro intophage particles producing a library of 5×10⁶ recombinant phageparticles.

Synthetic Oligonucleotides. Oligonucleotide probes complementary toregions of the gene encoding the 7,000 dalton peptide component of theTA4 antigen were synthesized using a Biosearch Sam I (Biosearch, Inc.,San Rafael, Calif.). The expected DNA sequences of the appropriateregions were deduced from the amino acid sequence of the 17,000 daltonpeptide. Because of the ambiguity in the genetic code, the exact DNAsequence cannot be predicted. "Mixed probes" were designed andsynthesized which contained a mixture of DNA sequences, one of whichshould have perfect match homology with the gene for the 17,000 daltonpeptide.

Oligonucleotide COD 92 was based on amino acids 6 to 12 of peptide V1(see Example 5 for amino acid sequence of the 17,000 dalton peptide). Itcontained a mixture of 256 different sequences. The structure ofoligonucleotide COD 92 is: ##STR2## Oligonucleotide COD 94 was based onamino acids 3 to 8 of peptide V2 of the 17,000 dalton peptide. Itcontained a mixture of 64 different sequences: ##STR3## OligonucleotideCOD 108 was based on amino acids 25-30 of peptide V1. It contained amixture of 16 different sequences. The structure of oligonucleotideCOD-108 is: ##STR4##

Screening the E. tenella Genomic DNA Library. Recombinant phage of theE. tenella genomic DNA library were plated on 15 cm plates at highdensity, up to 2-3×10⁴ phage per plate. Nitrocellulose filter replicasof each plate were prepared according to the method of Benton and Davis(3). The filters were then incubated with the appropriate syntheticoligonucleotides which had been labeled to high specific activity with(³² P)-dATP and T4 polynucleotide kinase. Positive plaques wereidentified by autoradiography. Only those plaques that hybridized toboth oligonucleotides COD-92 and 108 were scored positive.

Small blocks of agar were cut from the plates in regions thatcorresponded to the region of the filter containing the hybridizing DNA.The phage were eluted, replated at lower density (20-100/plate) andrescreened with all three oligonucleotide probes. Pure isolated positiveplaques or clones were picked. Phage 108-1 hybridized strongly tooligonucleotide COD-92 and moderately to oligonucleotides COD-108 and94. Phage 108-1 was grown up on a larger scale for purification andcharacterization of the E. tenella DNA insert. Characterization of phage108-1 DNA showed an EcoRI insert of 5,500 bp.

Detailed Characterization of the Genomic Clone Encoding the 17,000Dalton Peptide--Restriction Map. The 5,500 bp EcoRI fragment insert ofclone 108-1 was subcloned from the phage vector into plasmid pUC 9 (78).The recombinant plasmids were digested with a variety of restrictionendonucleases to determine the position of key restriction sites in thegenomic DNA clone. The position of restriction sites within the DNA wasneeded to determine the location and orientation of the 17,000 daltonpeptide gene and to develop a strategy to sequence the EcoRI genomic DNAfragment. The restriction map is presented in FIG. 2. The location andorientation of the gene for the 17,000 dalton peptide is shown on thismap.

DNA Sequence Analysis of Clone 108-1. The BglII-EcoRI fragment of clone108-1 containing the gene for the 17,000 dalton peptide component of theTA4 antigen was sequenced by the dideoxy method of Sanger (62) usingvarious restriction enzyme fragments. Primers for DNA synthesis includedoligonucleotides COD-92, 94 and 108 as well as other syntheticoligonucleotides. The DNA sequence is shown in FIG. 5.

Structure of the Gene Encoding the TA4 Antigen. The DNA sequence agreeswith that predicted by the amino acid sequence analysis. In addition,there are three features of the gene which are not apparent from theprotein sequence. Using protein sequence information and generalinformation regarding the structure of secretory proteins, the structureof the gene for the TA4 antigen has been deduced.

From the known amino terminus of the sporocyst membrane 17,000 daltonpeptide (see Example 5), Gln-Asp-Tyr---, it is apparent that the geneencodes an extra 23 amino acids upstream. This DNA sequence is a typical"signal" sequence found at the amino terminus of genes for manysecretory or membrane proteins (4, 34). The peptide it encodes isrequired for the export of protein from their site of synthesis (thecytoplasm) to and/or through the plasma membrane. The signal peptide isusually removed during the secretory process. It is not surprising thatthe TA4 antigen is made with a signal peptide since it most likelytraverses the cytoplasmic membrane in order to be found at the outersurface of the sporozoite. The amino terminus of the signal sequence isassumed to be the Met codon since, essentially, synthesis of allproteins begin with methionine.

There are three regions of the gene in which the DNA sequences do notcoincide with the protein sequence. The first is a 101 bp segmentoccurring within the codon for Val-7 of the known mature 17,000 daltonprotein sequence. The second is a 114 bp sequence between the codons forGly-65 and Gly-66 of the 17,000 dalton peptide. The third is a 124 bpsequence within the codon for Asp-186 of the 8,000 dalton peptide. Thesethree sequences are intron structures typically found within the codingregions of many eukaryotic genes. They are present in the precursor tothe mRNA, and then removed by an RNA recombination mechanism known as"splicing," to give the mature mRNA an uninterrupted coding sequence.The DNA sequences around the "splice junctions" are consistent withthose seen in other eukaryotic genes (65).

The sequence of the 17,000 dalton peptide appears to terminate with thesequence Gly-Gly corresponding to codons 157 and 158. We have alsoidentified an 8,000 dalton peptide with the sequence beginning withAla-162 extending to Glu-188. The peptide sequence Arg-Arg-Leucorresponding to codons 159 through 161 has not been found. It isprobable that this tripeptide is removed by a mechanism similar to thecleavage of other proteins such as insulin (71). Hence the two peptidesof the TA4 antigen are encoded by a contiguous nucleotide sequence, andat least one proteolytic step occurs to generate the 8,000 daltonpeptide beginning with Ala-162.

EXAMPLE 7

Appearance of the TA4 Antigen During Sporulation

In order to determine when in the process of sporulation the TA4 antigenoccurs, its appearance was measured by immunoreaction with a specificmonoclonal antibody, Ptn 9.9 D12. Monoclonal antibody Ptn 9.9 D12 is asporozoite-neutralizing monoclonal antibody that reacts with the TA4antigen. Reducing conditions destroy the reactivity of the TA4 antigenwith monoclonal antibody Ptn 7.2 A4/4. However, on Western blots of SDSPAGE under reducing conditions monoclonal antibody Ptn 9.9 D12 reactswith the 17,000 dalton polypeptide component of the TA4 antigen.

Starting immediately after the final PBS wash (see Example 1) aliquotscontaining 1×10⁷ oocysts were removed for analysis at four hourintervals up to 24 hours and at 36 to 48 hours after sporulation wasbegun. Sporulating oocysts were centrifuged at 7-800×g for 10 minutesand the supernatant was removed. The pellets were quick-frozen in a dryice/methanol bath and then stored at -70° C. until analysis.

Each pellet was thawed in 200 microliters of 20 mM Tris-HCl pH 7.5, 50mM MgCl₂, 25 mM NaCl and an equal volume of glass beads. After shakingvigorously for 10 minutes 200 microliters of 2 x SDS PAGE sample buffer(35) was added. Samples were boiled for 3 minutes, centrifuged to removedebris and 25-50 microliters of each sample was applied to SDSpolyacrylamide gels (5-25% gradient) for analysis. Proteins weretransferred to nitrocellulose sheets (5, 75). The remaining proteinbinding sites on the nitrocellulose were blocked with 3% (w/v) gelatin,10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (w/v) NaN₃ for 30 minutes atroom temperature. Nitrocellulose filters were incubated with monoclonalantibody Ptn 9.9 D12 (approximately 10 micrograms/ml in 3% (w/v) bovineserum albumin, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (w/v) NaN₃)overnight at 4° C. After washing the nitrocellulose filters three timeswith 50-100 ml of the antibody dilution buffer, the location and amountof bound monoclonal antibody Ptn 9.9 D12 was determined using theVectastain ABC Kit for mouse IgG (Vector Laboratories, Inc., Burlingame,Calif.). Nitrocellulose filters were immersed in 20 ml of biotinylatedhorse anti-mouse IgG (80 microliters biotinylated anti-mouse antibody,80 microliters normal horse serum in 20 ml antibody dilution buffer) andgently shaken for 30 minutes at room temperature. Nitrocellulose filterswere rinsed three times with 50-100 ml of antibody dilution bufferwithout NaN₃ Nitrocellulose filters were then incubated with 15 ml ofVectastain ABC Reagent for 30 minutes at room temperature (80microliters Avidin DH Reagent A mixed with 80 microliters biotinylatedhorseradish peroxidase Reagent B in 15 ml antibody dilution bufferwithout NaN₃ preincubated for 30 minutes before addition to thefilters). After three washes bound horseradish peroxidase was measuredby color development with 4-chloro-1-napthol (Sigma Chemical Co., St.Louis, Mo.). Blots were incubated with the color development solution (2ml of 3 mg 4-chloro-1-napthol/ml methanol, 5 microliters 30% hydrogenperoxide in 10 ml 10mM Tris-HCl pH 7.5, 150 mM NaCl) for 10-30 minutes.After appearance of the purple bands indicating the location and amountof Ptn 9.9 D12 reactive material, the nitrocellulose sheets were washedtwice with water, air dried and stored in the dark.

The 17,000 dalton polypeptide component of the TA4 antigen that wasimmunoreactive with monoclonal antibody Ptn 9.9 D12 appeared between 16to 24 hours after the initiation of sporulation and thereafter (FIG. 4).Sixteen hours corresponds with the beginning of elongation of the fourstructures destined to become sporocysts inside the sporulating oocyst.

EXAMPLE 8 Isolation and Identification of mRNA Encoding the TA4 Antigen

Before cDNA, could be synthesized it was necessary to determine when themRNA encoding the TA4 antigen appeared during sporulation. Aliquotscontaining 2.5-5×10⁸ oocysts were asceptically removed at four hourintervals up to 24 hours (including time 0) and at 36 to 48 hours aftersporulation was begun. The sporulating oocysts were centrifuged at7-800×g for 10 minutes and the supernatant was removed. The pellets werequick-frozen in a dry ice/methanol bath and then stored at -70° C. untilRNA was isolated.

Each pellet was thawed in approximately 10 volumes of 5M guanidinethiocyanate, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 5% (v/v)beta-mercaptoethanol and oocysts were rapidly broken by shakingvigorously with an equal volume of 1.0 mm glass beads for 10 minutes.After bringing the samples to 2% (w/v) N-lauroylsarcosine they werecentrifuged at approximately 8,000×g at room temperature to removedebris. RNA was isolated from the supernatant by sedimentation through aCsCl cushion (76).

The RNA pellet was resuspended in 20 mM Tris-HCl pH 7.5, 50 mM EDTA pH8.0, 0.2% SDS, 100 units/ml RNasin™ (Promega Biotec, Madison, Wisc.), 10mM beta-mercaptoethanol. After extracting twice alternately withphenolchloroform:isoamyl alcohol (24:1) and chloroform:isoamyl alcohol(24:1) the RNA was precipitated and stored in ethanol at -20° C.Approximately 100-300 micrograms of total RNA was isolated from2.5-5.5×10⁸ oocysts.

PolyA-containing RNA was isolated by oligo-dT cellulose chromatography(2). Total RNA was loaded on an oligo-dT cellulose column (Type 3,Collaborative Research, Inc., Lexington, Mass.) in 10 mM Tris-HCl pH7.5, 1 mM EDTA, 0.2% (w/v) SDS, 0.4M LiCl. RNA was eluted at 40° C. inthe same buffer without LiCl. Approximately 5-15 micrograms A⁺ RNA wasisolated from 2.5-5.0×10⁸ oocysts.

Before polyA RNA could be used as a template for cDNA synthesis, it wasnecessary to demonstrate the presence of the mRNA encoding the TA4antigen. The presence of the TA4 antigen mRNA was demonstrated byhybridizing polyA RNA from oocysts at various stages of sporulation withDNA from the clone encoding the TA4 protein. Two micrograms of polyA RNAfrom each time point during sporulation was electrophoresed through gelscontaining formaldehyde (44). The RNA was transferred to nitrocellulosefilters for Northern blot analysis. Nitrocellulose filters were probedwith the 785 bp SacI-PvuII fragment of the E. tenella genomic clone108-1 (FIG. 5) which had been nick translated with [³² P]-dATP (44). ThemRNA encoding the TA4 antigen was present approximately 16-20 hoursafter sporulation was initiated and thereafter (FIG. 6). The time ofappearance of the mRNA for the TA4 antigen correlates exactly with theappearance of the 17,000 dalton subunit of the TA4 antigen that isimmunoreactive with monoclonal antibody Ptn 9.9 D 12 on Western blots.These experiments demonstrate that mRNA from sporulated oocysts could beused to make cDNA encoding the TA4 antigen.

EXAMPLE 9 Isolation and Characterization of a cDNA Clone Encoding theTA4 Antigen

cDNA

The nucleotide sequence encoding the TA4 antigen was to be used as agene in an easily grown cell such as E. coli to produce a TA4 proteinfor vaccination of chickens against coccidiosis caused by certainEimeria. There are three regions of the TA4 gene (FIG. 5) in which theDNA sequence does not coincide with the protein sequence. These threesequences are introns typically found within the coding regions of manyeukaryotic genes. However, since genes containing introns would notexpress the proper protein in E. coli it was necessary to isolate a cDNAclone encoding the TA4 antigen. This clone contains a continuous codingsequence for the TA4 antigen.

Synthesis of cDNA

Briefly, the sporulated oocyst mRNA isolated as described in Example 8was transcribed into cDNA by the action of AMV reverse transcriptase asdescribed by Ullrich et al. (76). Transcription was initiated at the3'-polyadenylated end of the TA4 antigen mRNA using oligo-dT as aprimer. The second DNA strand was copied using DNA Polymerase I (theKlenow fragment). From 2 micrograms of mRNA we obtained 340 ng cDNA.

Specifically, 2 micrograms of oligo-dT (12-18 nucleotides, PharmaciaMolecular Biology Division, Piscataway, N.J.) was annealed to 2micrograms of purified mRNA in the presence of 50 mM NaCl. The annealingreaction was heated to 90° C. and then slowly cooled. For the reversetranscriptase reaction, deoxynucleosidetriphosphates (A, T, G, C) wereadded to 0.5 mM along with 40 units of enzyme (Molecular GeneticResources, Tampa, Fla.). The reverse transcriptase reaction buffer wasas follows: 15 mM Tris-HCl, pH 8.3, 21 mM KCl, 8 mM MgCl₂, 0.1 mM EDTA,and 30 mM beta-mercaptoethanol. This mixture was incubated at 42° C. for45 minutes. The RNA-DNA duplex was extracted once with phenol chloroformand then precipitated with ethanol. The pelleted material was thenresuspended in 100 microliter reaction mixture containing the following:20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 100 mM KCl and 250 mM each dATP,dCTP, dTTP, dGTP.

RNAse H (100 units/ml Pharmacia Molecular Biology Division, Piscataway,N.J.) and DNA Polymerase I--Klenow fragment (50 units/ml BoehringerMannheim, Indianapolis, Ind.) were added and the reaction was incubatedat 12° C. for 60 minutes. The combined activities of these enzymesresult in the displacement of the mRNA from the RNA-DNA duplex as thefirst cDNA strand is used as a template for synthesis of the second cDNAstrand. The reaction was stopped by the addition of EDTA to a finalconcentration of 10 mM and the DNA duplex was then extracted withphenol:chloroform and ethanol precipitated. The sequence of thereactions of DNA Polymerase I and RNAse H was predicted to yield cDNAmolecules which were blunt ended at both their 3' and 5' ends. A 3'blunt end was necessary for the subsequent cloning of the cDNA.

Construction of the TA4 cDNA Library

The cDNA was resuspended in 100 microliters of sterile water. To clonethe cDNA into a library a restriction site was used that had beendetermined from the genomic clone 108-1 DNA sequence. A SacI site isimmediately upstream to the N-terminal glutamine of the mature 17,000dalton subunit of the TA4 antigen. Approximately 50 ng was digested withSacI (50 units/ml) in the presence of 6 mM Tris-HCl (pH 7.4) 6 mM MgCl₂,and 6 mM beta-mercaptoethanol for 60 minutes at 37° C.

The sample was then re-extracted with phenol:chloroform and ethanolprecipitated. For the cloning step a pUC18 vector (56) was used. Thevector had been digested with SacI and SmaI. SmaI provided the blunt endsite necessary for ligation of the 3' end of the cDNA. The ligationreaction was performed using 40 ng of vector DNA and 50 ng of cDNA.Ligations were done overnight at 12° C. in a ligase buffer of 50 mMTris-HCl (pH 7.8), 10 mM MgCl₂, 20 mM dithiothreitol, 1.0 mM rATP usingone unit of T4 DNA ligase.

The recombinant DNA molecules were then introduced into Escherichia coliK-12 strain MH1 by transformation. The transformed bacteria were spreadon agar plates containing the antibiotic ampicillin at a concentrationof 50 micrograms/ml. Since the plasmid pUC18 (56) contains theampicillin resistance gene, only those bacteria which acquired arecombinant plasmid survived. These bacteria each grew and divided toform a bacterial colony. Each cell in the colony is a descendant of theoriginal parental cell and contains the same recombinant plasmid.Approximately 6700 clones were obtained from the 55 nanograms of cDNAused to make recombinant plasmids.

Identification of TA4 cDNA Clones

This cDNA library was screened by colony hybridization using the highdensity screening method described by Grunstein and Hogness (20). The785 bp SacI-PvuII fragment of the genomic clone was purified and labeledwith ³² P by nick-translation (44). Positive clones were identified,purified and plasmid DNA was isolated for further analysis. Restrictionanalysis of the positive cDNA clone agreed with the map of the genomicclone. The cDNA insert of the clone designated as pTCD26 was sequencedby dideoxy sequencing using oligonucleotide primers made to correspondto the genomic clone (62). The sequence of the cDNA pTCD26 clone isshown in FIG. 7. This cDNA clone was transformed into an E. coli strainJM83, and the strain designated as JM83/pTCD26 was deposited with theAmerican Type Culture Collection, Rockville, Md., and assigned ATCCaccession No. 53315. This deposit was made pursuant to the BudapestTreaty On.

The DNA sequence agreed with that predicted from the genomic clone. Thepredicted amino acid sequence from the cDNA agreed with the TA4 antigenamino acid sequence obtained by protein microsequencing.

EXAMPLE 10 Expression of the cDNA Derived TA4 Antigen Gene in E. coli

Construction of cDNA Derived TA4 Direct Expression Plasmids

The cDNA clone provides the gene for synthesis of the TA4 protein inbacteria. However, the cDNA does not contain the proper signals to allowtranscription and translation in E. coli. Therefore, the cloned cDNA wasinserted into expression vectors that contain a strong promoter(s) forRNA polymerase and a ribosome binding site to initiate protein synthesisin E. coli upstream of the inserted cDNA. As used herein, the phrase TA4protein refers to the expression product of the cDNA of FIG. 7 or anyrecombinant TA4-derived material produced in a bacterial host cell. Thephrase TA4 antigen refers to the naturally-occurring material asexpressed by the genomic TA4 DNA, as present on the surface of thesporozoite or purified away from sporozoites.

Expression vectors pWHA33 and pWHA63 were constructed so that genescould be inserted in them to obtain expression in E. coli. Othersuitable plasmids known to one skilled in the art could also be used.Plasmids pWHA33 and pWHA63 are two examples of suitable plasmids. ThepWHA33 plasmid contains three promoters (lac, lambda P_(R) and trp) eachof which could direct transcription of an inserted gene. Plasmidscontaining various combinations of these promoters and the tac promoterfrom plasmid pDR450 (61) (Pharmacia) Molecular Biology Division,Piscataway, N.J.) were constructed. The structure of plasmids pWHA33 andpWHA63 are diagrammatically shown in FIG. 8.

One strategy to synthesize the TA4 protein in E. coli is to simplyprovide a ribosomal binding site and a methionine codon (ATG) precedingthe coding sequence. To construct such a direct expression plasmid forthe TA4 protein, the cDNA clone pTCD26 was digested with SacI and thentreated with Klenow fragment of DNA polymerase I to produce blunt ends.An oligonucleotide linker COD-154 was ligated to the blunted SacI end toprovide the ATG codon to initiate protein synthesis and the BamHI sitenecessary to clone into the BamHI site of pWHA63. The structure ofCOD-154 is: ##STR5##

The insertion of TT immediately preceding the initiation codon ATG inCOD-154 is to improve efficiency of translational initiation.

After ligation of oligonucleotide COD-154 to the blunt ends of pTCD26,the product was digested with BamHI. The 1276 bp fragment containing theTA4 gene was purified on a polyacrylamide gel and then ligated into theBamHI site of expression vector pWHA63 resulting in plasmid pDET1. Theconstruction of pDET1 is diagramatically shown in FIG. 8. Another directexpression plasmid, pDET2 was constructed from pDET1 by digestion ofpDET1 with HindIII and religation which removed the HindIII fragmentcontaining lambda P_(R) and lambda cI. The pDET1 and pDET2 directexpression vectors were transformed into E. coli strain REN3.

The recombinant DNAs and host microorganisms described herein asREN3/pDET1 and REN3/pDET2 were deposited with the American Type CultureCollection, Rockville, Md. and assigned ATCC Accession Numbers 53316 and53318, respectively. These deposits were made pursuant to the BudapestTreaty.

Synthesis and Analysis of Cloned Gene Products in E. coli

Lysates of cells containing pDET1 and pDET2 were analyzed for thepresence of the TA4 protein. Proteins synthesized by the pDET1 and pDET2DNA were identified by probing Western blots of cell lysates with mouseantiserum raised against the reduced, denatured 17,000 dalton subunit ofthe E. tenella TA4 antigen.

Expression of pDET1 and pDET2 was analyzed first in E. coli strain W3110(W3110 carries the wild-type Lon+ protease gene). Cultures ofW3110/pDET1 and W3110/pDET2 were grown in L-broth (10 g/l tryptone(Difco), 5 g/l yeast extract (Difco), 5g/l NaCl, adjusted to pH 7.5 withNaOH) supplemented with 100 micrograms/ml ampicillin. To obtain optimumexpression, cultures were shaken at 30° C. to a cell density of 1-5×10⁸/ml, diluted 1:5 into fresh media and shaken at 37° for 2 to 6 hours.Cells were harvested by centrifugation, washed in M9 salts (6 g/l Na₂HPO₄, 3 g/l KH₂ PO₄, 0.5 g/l NaCl, 1 g/l NH₄ Cl) and resuspended at5×10⁹ /ml in Laemmli gel sample buffer (35). Twelve microliter sampleswere heated 10 minutes, 100° C., and run on 121/2% SDS-PAGE, and eitherstained with Coomassie Blue, or transferred to nitrocellulose sheets andprobed with a 1:1000 dilution of mouse antiserum to reduced-denatured17,000 dalton TA4 polypeptide as described above.

Expression of the TA4 gene in pDET1 and pDET2 is very low in E. colistrain W3110. The 25,000 dalton TA4 protein is visible only faintly onWestern blots and not visible above background on Coomassie Blue stainedpolyacrylamide gels of total cell lysates, suggesting that net synthesisis less than 0.5% of total E. coli protein.

It appeared likely that the apparent low expression of pDET1 and pDET2was due to instability of the TA4 protein in E. coli W3110. Othereukaryotic proteins have been shown to be unstable when synthesized inE. coli (18) Therefore, plasmids pDET1 and pDET2 were transformed intoE. coli strain MTM6, deficient in the lon protease (7). MTM6 is anon-mucoid derivative of Lonstrain AB1899 (CGSC #1899).

Expression of the TA4 gene in pDET1 and pDET2 is greatly increased instrain MTM6 (Lon⁻) Expression was analyzed as described above for W3110.FIG. 9 compares synthesis of pDET1 in W3110 (Lon⁺) and MTM6 (Lon⁻).Strains containing either pDET1 pDET2 DNA produced a 25,000 daltonpolypeptide that is immunoreactive with the mouse serum made against thereduced, denatured E. tenella TA4 antigen. These results suggest thatwhereas the 25,000 dalton protein encoded by the TA4 antigen gene iscleaved in E. tenella to a 17,000 dalton and 8,000 dalton polypeptidelinked by a disulfide bond, this post-translational processing does notoccur in E. coli.

When the lysates were separated into soluble and insoluble components bycentrifugation, the vast majority of the 25,000 dalton protein waslocalized in the insoluble fraction of the cell lysate (FIG. 9). Thisinsoluble protein does not appear immunoreactive with monoclonalantibody Ptn 7.2 A4/4 which recognizes the TA4 antigen in sporozoitemembranes or extracted from sporozoite membranes without reduction ofdisulfide bonds.

Because the expression levels of pDET1 and pDET2 are very low in Lon⁺ Ecoli and because Lon⁻ E. coli might be impractical to grow in largescale cultures, the TA4 protein was stabilized by fusion to otherproteins. Any suitable protein could be utilized for this proteinfusion. The following examples illustrate only two of the possibleproteins which are suitable; namely betagalactosidase and prochymosin.

EXAMPLE 11 Expression of the TA4 Protein as a Beta-Galatosidase FusionProtein in E. coli

Construction of Beta-Galactosidase-TA4 Expression Plasmids

The observation that the greatest yield of the genetically engineeredTA4 protein was obtained in a protease deficient strain suggests thatthe TA4 protein is subject to degradation in normal E. coli cells. TA4gene fusion plasmids were constructed because attachment of the TA4protein to a large protein can stabilize it in E. coli. Severaleukaryotic proteins are more stable in bacteria as fused proteins (17,27). Recombinant plasmid pBGC23 is a hybrid constructed for expressionof a beta-galactosidase-TA4 antigen fusion protein. It was derived froma plasmid pDK2 which contains the lac regulatory region and virtuallythe whole beta-galactosidase gene (1008 codons) from lambda plac (22,63) inserted into the EcoRI site of plasmid pBR328, and from the cDNAclone pTCD26. The construction of pBGC23 is diagramatically shown inFIG. 10. Suitable plasmids other than pDK2 can also be used. PlasmidpDK2 is one example of a suitable plasmid.

The 1276 bp EcoRI-BamHI fragment from pTCD26 containing the TA4 cDNAsequence was cloned into plasmid pDK2 that had been digested with EcoRIand BamHI to generate plasmid pDK22. Clone pDK22 contained the expectedplasmid in which the TA4 cDNA sequence was fused to the C-terminalregion of the beta-galactosidase coding sequence. However, in thisplasmid the cDNA derived TA4 coding sequence is not in phase with thatof beta-galactosidase. Therefore, plasmid pDK22 was digested with EcoRIand then treated with DNA polymerase I Klenow fragment and religated toput the TA4 coding sequences into the proper reading frame. Thisplasmid, designated pBGC23, contains a hybrid gene that encodes aprotein consisting of the TA4 protein fused to the C-terminal region ofbeta-galactosidase (lacZ). pBGC23 was used to transform E. coli strainsJM83, and REN3.

The recombinant DNA and its host microorganism described herein asREN3/pBGC23 was deposited with the American Type Culture Collection,Rockville, Md. and assigned ATCC Accession Number 53317. This depositwas made pursuant to the Budapest Treaty.

Expression and Analysis of Cloned Gene Products

Proteins encoded by the pBGC23 DNA were identified by probing Westernblots of cell lysates with mouse serum against the purified reduceddenatured 17,000 dalton subunit of the E. tenella TA4 antigen asdescribed in Example 10. JM83/pBGC23 and REN3/pBGC23 were grown inL-broth, supplemented with 0.1% glucose and 100 micrograms/mlampicillin. Cultures were grown to saturation by shaking at 37° C.overnight. Cells were harvested by centrifugation, washed in M9 saltsand resuspended at 5×10⁹ /ml in Laemmli gel sample buffer. 20 microlitersamples were heated 10 minutes at 100° C. and run on 7-1/2% SDS-PAGE,and either stained with Coomassie Blue or Western blotted.

Stained and immunoblotted gels (FIG. 11) demonstrated that the expected135,000 dalton fusion protein is synthesized in JM83/pBGC23 andREN3/pBGC23 cultures but not in JM83 alone. The Western blot shows thatthe protein is immunoreactive with the mouse serum against the reduced,denatured E. tenella TA4 antigen. FIG. 11 shows the protein is presentin the insoluble fraction of the cell lysate. Cultures grown asdescribed above were lysed by sonication following lysozyme and EDTAtreatment, and cell membranes were solubilized in 2% Triton overnight at4° C. The insoluble material was collected by centrifugation, and the135,000 dalton pBGC23 product was present in this fraction.

The pBGC23 protein is synthesized in E. coli at high levels, but isinsoluble and does not react with monoclonal antibody Ptn 7.2 A4/4.Furthermore, this insoluble pBGC23 protein, when injected into mice willnot raise antibodies that cross-react with native TA4 antigen.

EXAMPLE 12 Expression of the TA4 Protein as a Prochymosin Fusion Proteinin E. coli

The proteins made by cells containing pDET1, pDET2 or pBGC23 are largelyor totally insoluble, and thereby are apparently not active withmonoclonal antibody Ptn 7.2 A4/4. It was observed that other eukaryoticproteins that are made in E. coli in an insoluble, inactive form can besolubilized and their activity recovered. One such protein is bovineprochymosin. The TA4 cDNA sequence was fused to the bovine prochymosingene to produce an insoluble fusion protein that could be solubilizedand made active by procedures developed for prochymosin alone. Theextent of proper renaturation of the fusion protein could then bemonitored by following chymosin activity.

A plasmid-encoded prochymosin-TA4 fusion protein was created by joiningthe TA4 cDNA sequence to the cloned bovine prochymosin gene of pWHA43,which directs synthesis of prochymosin in a stable but insoluble form inE. coli (47). Other plasmids may also be utilized. One suitable plasmidis pWHA43.

In order to construct the prochymosin-TA4 gene fusion, pWHA43 wasconverted to pWHA93 as shown in FIG. 12. First, the tac promoter ofpDR540 (61) was substituted for the trp promoter to produce pWHA49 byspecific restriction endonuclease substitution. Next, the normal stopcodon of prochymosin was removed by substituting the C-terminal portionof the prochymosin gene of pWHA49 with a modified prochymosin C-terminalportion from pMH22, to give pWHA93. A spontaneous deletion resulted inthe removal of the tac promoter from pWHA93. pMH22 contains theC-terminal half of the gene from the cDNA clone p5G3, fused to theprochymosin Bc1I site (deleting the stop codon) to the polylinker andbeta-galactosidase gene fragment in plasmid pUC18.

In the construction of prochymosin-TA4 gene fusion, pCOC12, a 1294 bpfragment was removed from the cDNA clone pTCD26 by digestion with theenzymes EcoRI and PstI, followed by digestion with Mung bean nuclease toform blunt-ended DNA. The plasmid pWHA93 was digested with XbaI andtreated with Mung bean nuclease and the blunt-ended vector was ligatedwith the blunt-ended fragment containing the TA4 cDNA sequences (1286 bpafter Mung bean nuclease treatment) to generate the recombinant plasmidpCOC11. After this ligation, the TA4 derived sequences were found to beout of reading frame with the coding sequences of prochymosin. In orderto change the reading frame, pCOC11 was digested with SacI and Mung beannuclease, and was then religated to generate pCOC12. The construction ofpCOC12 is diagrammatically shown in FIG. 13. Plasmid pCOC12 was modifiedto pCOC14 by removal of two NarI fragments by NarI digestion andreligation, reducing the size of the plasmid but not deleting any of theprochymosin or TA4 sequences. Plasmid pCOC14 was modified to form pCOC20by removal of a 249 bp SphI fragment by digestion with SphI andreligation. The 249 nucleotide deletion in the prochymosin sequence ofpCOC20 maintains the correct reading frame, and results in an 83 aminoacid deletion in the prochymosin portion of the fusion protein.

For expression studies, pCOC12 and pCOC20 were transformed into strainREN3, a bacteriophage T1 resistant derivative of CY15001, a trp Rderivative of W3110.

The recombinant DNAs and host microorganisms described herein asREN3/pCOC12 and REN3/pCOC20 were deposited with the American TypeCulture Collection, Rockville, Md. and assigned ATCC Accession Numbers53314 and 53313, respectively. These deposits were made pursuant to theBudapest Treaty.

Expression and Analysis of Cloned Gene Products

Proteins encoded by the pCOC12 and pCOC20 DNAs were identifiedimmunologically, following fractionation by electrophoresis and transferto nitrocellulose sheets as described in Example 10.

REN3/pCOC12 and REN3/pCOC20 were grown to saturation in L-brothsupplemented with 0.1% glucose and 100 micrograms/ml ampicillin byshaking at 30° C. overnight Cells were harvested by centrifugation,washed in M9 salts and resuspended in Laemmli sample buffer. Sampleswere heated 10 minutes, 100° C. and run on 10% polyacrylamide gels inSDS and either stained with Coomassie Blue or transferred tonitrocellulose sheets for immunologic analysis, as described.

Triton insoluble material was prepared from REN3/pCOC12 and REN3/pCOC20cultures as described in Example 11, and run on polyacrylamide gels.

The stained gels and Western blots shown in FIG. 14 show that pCOC12 DNAencodes a polypeptide of the expected molecular weight, approximately65,600 daltons that is immunoreactive with the mouse serum against thereduced, denatured E. tenella TA4 antigen. As expected, the protein ispresent in the insoluble fraction of the cell lysate. Plasmid pCOC20 DNAencodes an immunoreactive polypeptide with the expected molecular weightof 56,500. The TA4 protein from pCOC20 is also present in the insolublefraction of the cell lysate.

EXAMPLE 13 EXTRACTION OF THE TA4 PROTEIN FROM THE INSOLUBLE STATE ANDDEMONSTRATION OF IMMUNOREACTIVITY WITH MONOCLONAL ANTIBODY Ptn 7.2 A4/4

The E. coli products of expression plasmids pDET1, pDET2, pBGC23,pCOC12, pCOC20 are all largely or totally insoluble. All can besolubilized by boiling in Laemmli sample buffer and react with mouseantiserum raised against the 17,000 dalton TA4 antigen subunit. However,none react with monoclonal antibody Ptn 7.2 A4/4 under these conditions.Therefore, it was necessary to solubilize and renature these E. colisynthesized proteins to produce antigens in a form that would react withmonoclonal antibody Ptn 7.2 A4/4 and therefore could possibly raiseneutralizing and protective antibody responses against E. tenella inanimals.

Extraction and Renaturation of Bacterially Produced TA4 Proteins

First the pCOC12 protein was solubilized and renatured by methods knownto solubilize and renature bovine prochymosin to produce active enzyme(47). This procedure produced pure soluble pCOC12 protein that possessedboth prochymosin activity (milk clotting after acid activiation tochymosin) and Ptn 7.2 A4/4 immunoreactivity. Conditions were optimizedfor recovery of immunoreactivity and are described below.

Plasmid pCOC12 was constructed, as described above, by fusing the 3' endof the coding sequence of bovine prochymosin to the 5' end of the codingsequence of the TA4 polypeptide. This plasmid was used to transform E.coli strain REN3 using standard techniques and ampicillin resistantcolonies were purified and used for culturing. An ampicillin resistantcolony from a freshly streaked agar plate was used to inoculate a 5 mlliquid culture containing L-broth and ampicillin at 100 micrograms/ml.The culture was grown for several hours at 37° C. with shaking untilcells had grown to an easily visible turbidity. The 5 ml culture wastransferred to a flask containing 1 liter of L-broth/ampicillinsupplemented with 0.1% glucose. This culture was grown at 30° C. withshaking to stationary phase. Cells were collected by centrifugation andstored frozen at -70° C. 10 g of frozen cell paste of E. coli strainREN3 containing pCOC12 were suspended in 100 ml of 25 mM TrisHCl pH 8,10 mM EDTA, 1 mg/ml lysozyme. After a short incubation, the lysed cellswere further disrupted by sonication. Prochymosin synthesized in E. colihas been shown to be completely insoluble in cell lysates, even in thepresence of non-ionic detergents which solubilize cell membranes andmembrane proteins. Partial purification of the pCOC12-encodedprochymosin-TA4 fusion protein was achieved by centrifugation of thecell lysate at 10,000×g for ten minutes, followed by an overnightdetergent extraction of the pelleted cell debris with a buffer solutioncontaining 2% Triton X-100 detergent (Sigma Chemical Co., St. Louis,Mo.). The pCOC12 fusion protein remained insoluble and was collected bycentrifugation.

This purification was improved slightly by additional washing of theinsoluble material with the solution containing 2% Triton X-100. ThepCOC12 prochymosin-TA4 protein was suspended in 6.3 or 12.6 ml of 10 mMsodium phosphate buffer at pH 7.5. The suspension is fully solubilizedby the addition of solid urea to a final concentration of 6-8 M in avolume of 10 or 20 ml, respectively.

The resultant clear solution was diluted into 100 or 50 volumes,respectively of 10 mM sodium phosphate buffer adjusted to pH 11.0 toachieve a final volume of 1000 mls. The solution was mixed thoroughlyand allowed to stand for 20 minutes at 15°-25° C. The pH of the solutionwas then titrated to pH 8.3 by addition of 0.2N HCl over a period of 3minutes.

The resultant solution was left at room temperature for one hour or moreprior to assay or storage. This solution contained approximately 100micrograms/ml of the 65,600 dalton protein which was 80-90% pure. Thesample was assayed for chymosin enzymatic activity or immunoreactivitywith monoclonal antibody Ptn 7.2 A4/4 as detailed below.

Assay of chymosin activity was a convenient way to monitor recovery ofproperly renatured protein. Recovery of immunoreactivity with Ptn 7.2A4/4 correlated well with recovery of chymosin activity from pCOC12protein, as measured by milk-clotting activity following acidactivation. Recovery of immunoreactivity of the pCOC12 protein isdescribed below and shown in FIG. 15.

The other TA4 proteins and protein fusions described above weresolubilized and renatured by the same or similar methods. Plasmid pCOC20was constructed, as diagrammed in FIG. 13, by a SphI deletion within theprochymosin component of the pCOC14 fusion protein. This deletionmaintained the correct reading frame within the gene fusion and had noeffect on the insolubility or subsequent solubilization or renaturationof the pCOC20 fusion protein. While the pCOC20 fusion protein maintainedimmunoreactivity of its TA4 epitope, the deletion-containing prochymosindomain could not be activated to a form having milk clotting activity.

Plasmid pCOC20 was used to transform E. coli strain REN3 that wascultured as described above. The insoluble pCOC20 prochymosin-TA4protein was isolated and renatured from 10 grams of frozen cell paste ofREN3/pCOC20 as described above.

Plasmid pBGC23 was constructed, as diagrammed in FIG. 10, by fusing the3' end of the coding sequence of E. coli beta-galactosidase to the 5'end of the coding sequence of the cDNA derived TA4 polypeptide. Thisplasmid was used to transform E. coli strain JM83 (cultured as describedabove). The beta-galactosidase-TA4 fusion polypeptide was found to beinsoluble within a cell lysate and was isolated and renatured from 10gms of frozen cell paste of JM83/pBGC23 by the methods described above.Plasmid pDET2 was constructed, as diagrammed above, so as to express theTA4 protein directly rather than as a fusion polypeptide. Optimal yieldof the pDET2 was achieved in a protease deficient E. coli strain MTM6.This strain was cultured as described above with the followingexception. When the 1 liter culture of cells grown at 30° C. reached anoptical density of 0.5 (Abs at 600 nm) the temperature was raised to 37°C. for 1 to 2 hours. The cells were harvested and stored frozen at -70°C.

10 grams of frozen cell paste of MTM6/pDET2 were lysed using the methodsdescribed above, and the Triton insoluble protein was isolated anddissolved in urea as described above. The solubilized protein wasrenatured by extensive dialysis against 10 mM sodium phosphate buffer,pH 8.5.

Immunoassay of Renatured Samples

The immunoreactivity of the renatured pCOC12, pCOC20, pDET2 and pBGC23proteins with monoclonal antibody Ptn 7.2 A4/4 was measured relative tothe TA4 antigen purified from E. tenella sporocysts. Each well of themicrotiter plate (Immulon I microELISA flat-bottom well plates, DynatechLaboratories, Inc., Alexandria Va.) was coated with 100 microlitersantigen diluted in 10 mM Na₂ HPO4, 150 mM NaCl, 0.01% (w/v) Zwittergent3-12, pH 8.0. For renatured samples, 1:10 to 1:1000 dilutions of theantigen were assayed. The purified E. tenella antigen was assayed inparallel at concentrations of 0.5-10 ng/well. Plates were coated withthe antigens by incubation with the antigen solutions for 1 hour at roomtemperature and then overnight at 4° C. Wells were emptied and thenwashed three times with phosphate buffered saline pH 7.2 containing0.02% (v/v) Tween-20 (PBST). The plates were treated with 3% (w/v)gelatin, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (w/v) NaN₃ for 30minutes at room temperature to block any remaining protein bindingsites. Plates were then incubated with 100 microliters of monoclonalantibody Ptn 7.2 A4/4 (30 micrograms/ml in 3% [w/v] bovine serumalbumin), 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (w/v) NaN₃) for 2hours at room temperature. After rinsing the wells three times withPBST, the bound monoclonal antibody Ptn 7.2 A4/4 was determined usingthe Vectastain™ ABC Kit for mouse IgG (Vector Laboratories, Inc.,Burlingame, Calif.). Each well of the plate was filled with 100microliters of biotinylated horse anti-mouse IgG (40 microlitersbiotinylated anti-mouse antibody, 80 microliters normal horse serum in10 ml PBST) and incubated 30 minutes at room temperature. Plates wererinsed three times with PBST. Plates were then incubated bated with 100microliters/well of Vectastain™ ABC Reagent for 30 minutes at roomtemperature (80 microliters Avidin DH Reagent A mixed with 80microliters biotinylated horseradish peroxidase Reagent B in PBSTpreincubated for 30 minutes before addition to the plates). After fivewashes with PBST bound horseradish peroxidase was measured by theaddition of 100 microliters substrate/well (0.1 mg/ml2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) in 50 mMcitrate/phosphate buffer pH 5.3, 0.015% (v/v) hydrogen peroxide). Plateswere incubated in the dark at room temperature. The absorbance at 414 nmwas measured 10-60 minutes after substrate addition in a TitertekMultiscan™ automatic plate reader (Flow Laboratories, Inc., McClean,Va.).

The relative immunoreactivities of the various renatured antigens (e.g.,encoded by pBGC23, pCOC12, pCOC20 and pDET2) were compared with that ofthe TA4 antigen extracted from E. tenella oocysts. Increasing amounts ofeach protein were added to microtiter plate wells and the OD₄₁₄ of thereaction in each well was plotted against the antigen mass present (FIG.15). The immunoreactivity of the bacterially-produced proteins subjectedto the denaturation/renaturation treatment described above range between10 and 30% of the activity of the E. tenella-extracted protein, on amolar equivalent basis.

EXAMPLE 14 Extraction of Immunoreactive Direct Expression TA4 Proteinfrom the Insoluble State

The E. coli products of expression plasmids pDET1, pDET2, pBGC23, pCOC12and pCOC20 are all largely or totally insoluble. All can be solubilizedby boiling in Laemmli sample buffer and react with mouse antiserumraised against the 17,000 dalton TA4 antigen subunit. However, nonereact with monoclonal antibody Ptn 7.2 A4/4 under these conditions.Therefore, it was necessary to solubilize and renature these E. colisynthesized proteins to produce antigens in a form that would react withmonoclonal antibody Ptn 7.2 A4/4 and therefore could possibly raisedneutralizing and protective antibody responses against E. tenella inanimals.

Solubilization and renaturation of the TA4 antigen-prochymosin fusionpCOC12 has been described.

Plasmid pDET2 was constructed, as diagrammed above, so as to express theTA4 protein directly rather than as a fusion polypeptide. Optimal yieldof the pDET2 was achieved in protease deficient E. coli strains MTM6 andSG936.

Culture conditions were optimized for recovery of antigen and aredescribed below.

The pDET2 protein was solubilized and renatured by methods known tosolubilize and renature TA4 prochymosin fusions to produceimmunoreactive antigen (Example 13). This procedure produced puresoluble pDET2 protein that possessed Ptn 7.2 A4/4 immunoreactivity.

Plasmid pDET2 was constructed, as described above. This plasmid was usedto transform E. coli strain SG936 using standard techniques andampicillin resistant colonies were purified and used for culturing. Anampicillin resistant colony from a freshly streaked agar plate was usedto inoculate a 100 ml liquid culture containing L-broth and ampicillinat 100 micrograms/ml. The culture was grown overnight at 30° C. withshaking. The 100 ml culture was transferred to a flask containing 1liter of L-broth/ampicillin. This culture was grown at 30° C. withshaking to OD₆₀₀ of 1.0. IPTG was added to 2 mM and the culture wasgrown 2-3 hours more at 30° C. Cells were collected by centrifugationand stored frozen at -70° C. 5 g of frozen cell paste of E. coli strainSG936 containing pDET2 were suspended in 40 ml of 25 mM Tris-HCl pH 8,10 mM EDTA, 0.5 mg/ml lysozyme. After a short incubation, the lysedcells were further disrupted by sonication. Because the pDET2 proteinsynthesized in E. coli has been shown to be completely insoluble in celllysates, pDET-encoded TA4 protein was purified by centrifugation of thecell lysate at 100,000×g for 1 hour, followed by a detergent extractionof the pelleted cell debris with a buffer solution containing 5% TritonX-100 detergent (Sigma Chemical Co., St. Louis, Mo.), 20 mM EDTA, for 60minutes at 25° C. The pDET2 protein remained insoluble and was collectedby centrifugation at 100,000×g.

The pDET2 insoluble material was suspended in 12 ml, 10 mM sodiumphosphate (pH 7.5) and collected by centrifugation to remove remainingdetergent. The pDET2 TA4 protein was suspended in 10 mM sodium phosphatebuffer at pH 7.5 to a final volume of 7.7 ml. The suspension is fullysolubilized by the addition of 5.8 g solid urea to a final volume of1200 mls. The solution was mixed thoroughly and allowed to stand for 10minutes at 150° C. The pH of the solution concentration of 8 M in avolume 12 ml, and then mixed for 16 hours at room temperature.

The resultant clear solution was diluted into 100 volumes of 10 mMsodium phosphate buffer adjusted to pH 11.0 to achieve a final volume of1200 mls. The solution mixed thoroughly and allowed to stand for 10minutes at 15° C. The pH of the solution was then titrated to pH 8.5 byaddition of 0.5N HCl over a period of 5 minutes.

The resultant solution was left at room temperature for one hour or moreprior to assay or storage. This solution contained approximately 10micrograms/ml of the 25,000 dalton protein which was 50-60% pure. Thesample was assayed for immunoreactivity with monoclonal antibody Ptn 7.2A4/4 as detailed in Example 13. The preparation had activity comparableto renatured antigens from pBGC23 and pCOC12 described above. Proteinwas concentrated to 2 mg/ml for the vaccination studies, below.

EXAMPLE 15 Use of E. tenella TA4 Antigen and an 11,500 Dalton FragmentThereof to Elicit Sporozoite Neutralizing Serum Response and ProtectiveResponse Against E. tenella in Chickens

Eliciting Sporozoite Neutralizing Serum Response Against E. tenellaUsing the TA4 Antigen. The TA4 antigen used in these experiments wasprepared from sporocysts by methods described in Example 4 for thepreparation of the nonreduced intact TA4 antigen. Purity and identity ofthe protein was confirmed by SDS-PAGE and immunoreactivity withmonoclonal antibody Ptn 7.2A4/4 prior to use in chickens.

Vaccine preparations were formulated at a level of one volume antigen tothree volumes of oil carrier consisting of about 5% Arlacel A, 94%Drakeol 6-VR, 1% Tween 80 so that each 0.1 ml dose containedapproximately 15 micrograms of TA4 antigen. When necessary, antigen wasdiluted with PBS (pH7.2) to the level desired for formulation. Chickensreceived 0.1 ml dose by intramuscular route in the neck muscle. Antigenwas administered two more times by the same route using the same amountat two-week intervals.

Three days prior to each administration of protein, and eleven daysafter the final administration, birds were bled for collection of serumsamples. Heat inactivated sera were tested independently in thesporozoite microneutralization assay as described in Example 2.Neutralization of parasites by serum was scored on the basis of themaximum serum dilution affording 50% inhibition of schizont development.The results as set forth below in Table I indicate that whereasnonvaccinated birds receiving carrier only had no demonstrableneutralizing antiserum titers against E. tenella sporozoites, birdsreceiving three doses of the antigen had demonstrable neutralizingantiserum titers.

                  TABLE I                                                         ______________________________________                                        TA4 Antigen Induced Sporozoite                                                Neutralization Assay Data                                                                 Sporozoite Neutralization                                                     Titers (ND 50%).sup.d                                             Serum Samples Highest   Lowest   Median Titer                                 ______________________________________                                        Prebleed.sup.a                                                                              .sup. N.D..sup.b                                                                        N.D.     N.D.                                         Nonvaccinate Controls                                                                       N.D.      N.D.     N.D.                                         (n = 9)                                                                       Carrier Only (n = 14)                                                                       N.D.      N.D.     N.D                                          Carrier/Protein                                                                             1:32      N.D.     1:8                                          Vaccine (n = 15)                                                              Immune serum.sup.c                                                                          --        --       1:32                                         (Whole Sporozoite                                                             Vaccinates                                                                    ______________________________________                                         .sup.a Serums from birds within each treatment group were pooled and          tested.                                                                       .sup.b N.D. = No detectable neutralization                                    .sup.c Pooled serum from several birds                                        .sup.d 50% neutralization dose                                           

Eliciting a Protective Response in Chickens Using the TA4 Antigen.Sixty-three (63) days after the final vaccination, some birds werechallenged, orally with 1,000 sporulated E. tenella oocysts. This wasfollowed the next day with 3,000 sporulated E. tenella oocysts alsogiven orally. Caecal lesions were scored 5 days after the finalchallenge. The results are tabulated below in Table II.

                  TABLE II                                                        ______________________________________                                        Protection of TA4 Antigen Vaccinated Birds                                    Against E. tenella Coccidiosis                                                                  Lesion Score .sup.--X ± S.D.                             ______________________________________                                        Nonvaccinate Controls (n = 17)                                                                    3.4 ± 0.6                                              Adjuvant Only (n = 5)                                                                             4.0 ± 0.0                                              TA4 Antigen/Adjuvant                                                                              2.4 ± 1.3                                              Vaccinates (n = 8)                                                            ______________________________________                                    

Eliciting Sporozoite Neutralizing Serum Response Against E. tenellaUsing the 11,500 dalton fragment of the TA4 Antigen. The immunogen usedin these experiments was prepared from sporocysts by phenol extractionas described in Example 4. Purity and identity of the protein wasconfirmed by SDS-PAGE and immunoreactivity with monoclonal antibody Ptn7.2A4/4 prior to use in chickens.

Lyophilized purified antigen was dissolved in 0.15 M phosphate bufferedsaline and emulsified in three parts carrier consisting of about 5%Arlacel A, 94% Drakeol 6-VR, 1% Tween-20 at a final antigenconcentration of 70 micrograms/ml. Chickens received about 14 microgramsprotein/0.2 cc dose by intramuscular route in the neck muscle. Antigenwas again administered two weeks later by the same route using the sameamount.

One day prior to each administration of protein, and two weeks after thesecond administration of protein, birds were bled for collection ofserum samples. Heat inactivated sera were tested independently in thesporozoite microneutralization assay as described in Example 2.

The results as set forth below in Table III indicate that whereasnonvaccinated birds receiving carrier only had no demonstrableneutralizing antiserum titers against E. tenella sporozoites, birdsreceiving two doses of antigen had demonstrable neutralizing antiserumtiters of up to 1:81.

                  TABLE III                                                       ______________________________________                                        Sporozoite Neutralizing Assay Data                                                     Sporozoite Neutralization Titers (ND 50%).sup.a                      Serum Sample*                                                                            Bleeding Highest  Lowest                                                                              Median Titers                              ______________________________________                                        Prebleed    0 Week.sup.                                                                           1:3      1:3   1:3                                        Nonvaccinate                                                                             2 Weeks  1:3      1:3   1:3                                        Controls   4 Weeks  1:3      1:3   1:3                                        Carriers only                                                                            2 Weeks  1:3      1:3   1:3                                                   4 Weeks  1:3      1:3   1:3                                        Carrier/Protein                                                                          2 Weeks  1:3      1:3   1:3                                        Vaccine    4 Weeks   1:81    1:3   1:9                                        Immune Serum**                                                                           --       --       --     1:81                                      (Whole Sporo-                                                                 zoite vaccine)                                                                ______________________________________                                         *5 birds per group                                                             **Pooled serum from several birds                                            .sup.a A 50% neutralizing dose                                           

Eliciting a Protective Response in Chickens Using the 11,500 DaltonFragment of the TA4 Antigen. Birds received approximately 3 microgramsof antigen in the aforementioned carrier one time in the neck muscle. Asecond group of birds received the carrier substance only. A final groupof nonvaccinate (sentinel) birds were housed with each of the twoaforementioned groups. Birds were exposed to coccidia by being housed inE. tenella contaminated cages. Approximately two weeks later, the birdswere examined and found to have been infected by E. tenella. The resultsare shown in Table IV below.

                  TABLE IV                                                        ______________________________________                                        Protection of Vaccinate Birds Against                                         Coccidiosis by E. Tenella                                                     Treatment    Lesion Score .sup.--X .sup.+  S.D.                                                            No. of Deaths                                    ______________________________________                                        Adjuvant only                                                                              3.8 + 0.4       2                                                (n = 5)                                                                       Antigen vaccination                                                                        1.0 ± 0.8    0                                                (n = 5)                                                                       Sentinal Birds                                                                             4.0 ± 0.0    6                                                (n = 6)                                                                       ______________________________________                                    

Because the conditions described above closely mimic the natural meansof exposure to E. tenella in the field, the data presented show clearevidence of the usefulness of the invention for protection againstcoccidiosis due to E. tenella.

Demonstration that Neutralizing Serum Antibodies of Chickens Recognizethe 17,000 Dalton Polypeptide Component of the TA4 Antigen. Analysis ofserum antibody specificity for the 17,000 dalton polypeptide componentof the TA4 antigen was performed using Western blots (7, 59). Allchicken sera with demonstrable neutralization titers to E. tenellasporozoites were shown to possess immunoglobulins with specificity forthe 17,000 dalton peptide component of the TA4 antigen; conversely, nosera from nonresponding or control birds had specificity for the 17,000dalton polypeptide or any other sporozoite protein.

Demonstration that Neutralization Serum Antibodies of Chicken CompeteWith Monoclonal Antibody Ptn 7.2A4/4. Sera from vaccinated birds withdemonstrable neutralization titers to E. tenella sporozoites, as well ascorresponding control sera were tested for the ability to compete withantibody Ptn 7.2A4/4 for binding sites on sporozoite membranes.Polystyrene 96 well clusters (Immulon II) were sensitized with 50microliters of sporozoite membrane proteins in 10 mM glycine bufferedsaline, pH 9.6, at a level of approximately 100 micrograms totalprotein/ml. Serial two-fold dilutions of sera were prepared in 0.15Mphosphate buffered saline with 0.0005% Tween-20 (PBs-Tw) containing a1:80 dilution of alkaline phosphatase conjugated to Ptn 7.2A4/4 and thentransferred to the sensitized plates at a final volume of 75microliters/well. After incubation at 37° C. for 30 minutes, the plateswere rinsed free of unreacted materials using PBS-Tw. Afterward,substrate consisting of the sodium salt of P-phosphonitrophenoldissolved in 1M diethanolamine buffer at a level of 1 mg/ml was added toeach well of the plate to a final volume of 100 microliters. Theresultant reaction product was monitored spectrophotometrically. Fromthe study, it was possible to ascertain that sera from birds respondingto the vaccination as evidenced by neutralization and immunoblots alsocontained antibody which competed with monoclonal antibody Ptn 7.2A4/4.This experiment provides direct evidence that antigen purified fromsporozoite membranes by either immunoaffinity chromatography usingmonoclonal Ptn 7.2A4/4 or conventional chromatography is capable ofstimulating an immune response in chickens to the epitope defined bymonoclonal Ptn 7.2A4/4.

EXAMPLE 16 Use of E. tenella Protein to Elicit Sporozoite NeutralizingSerum Response Against E. necatrix in Chickens

Heat inactivated sera from birds vaccinated with the 1,500 daltoncontaining preparation of the E. tenella TA4 antigen (Example 4) werepooled and tested in the neutralization assay (Example 2) substitutingembryonic porcine lung cells. The results are as listed in Table Vbelow.

                  TABLE V                                                         ______________________________________                                        Treatment         Neutralization Titer                                        ______________________________________                                        Non-immune chicken serum                                                                        1:6                                                         TA4 Antigen Vaccination                                                                         1:24                                                        E. tenella whole sperozoite                                                                     1:48                                                        immune serum                                                                  ______________________________________                                    

The data demonstrate the development of an elevated serum neutralizationtiter against E. necatrix when birds receive the purified 11,500 daltonfragment of the TA4 antigen. Because it has been previously demonstratedthat administration of the TA4 antigen results in the elevation of serumneutralizing titers to E. tenella, and that administration of the TA4antigen results in protection from E. tenella challenge, and since E.necatrix sporozoite neutralization titers are elevated by theadministration of TA4 antigen, one skilled in the art would predict thatprotection against E. necatrix challenge will also result fromadministration of the TA4 antigen.

EXAMPLE 17 Use of Bacterially Produced TA4 Proteins to Raise Serumantibodies in Mice That Cross React with the E. tenella TA4 Antigen

The immunogenicity of bacterially-produced TA4 protein was tested bysubcutaneous injections in CB6-F1 mice. Renatured pCOC12 and pBGC23proteins as well as insoluble proteins from these constructs weretested. Purified E. tenella TA4 antigen was used as a positive controland renatured prochymosin (from strain REN3 containing pWHA49) as anegative control. A group of 5 mice was injected for each antigen. Micewere injected twice at a 35 day interval and bled about 10 days aftereach injection.

For the E. tenella TA4 antigen, 10 micrograms was injectedsubcutaneously per mouse in a mixture of 3 parts antigen solution to 5parts complete Freund's adjuvant. (Final volume 200microliters/injection). Renatured pCOC12 and pBGC23 proteins orinsoluble proteins from these plasmids were similarly injected atapproximately a twofold molar excess of the bacterial TA4 protein ascompared to the E. tenella antigen.

Sera were assayed by the ELISA method described in Example 13.Microtiter plates were coated with 2ng of the purified E. tenella TA4antigen/well. The results of the assay with sera from the second bleedare shown in the Table VI below.

                  TABLE VI                                                        ______________________________________                                                          Absorbance-Blank (414 nm)*                                  Antisera Raised Against                                                                         (.sup.--X ± S.D.)                                        ______________________________________                                        Renatured Prochymosin                                                                           0                                                           Renatured pCOC12 Protein                                                                        0.31 ± 0.06                                              Insoluble pCOC12 Protein                                                                        0.01 ± 0.01                                              Renatured pBGC23 Protein                                                                        0.29 ± 0.05                                              Insoluble pBGC23 Protein                                                                        0.03 ± 0.04                                              E. tenella Purified TA4                                                                         0.36 ± 0.11                                              ______________________________________                                         *5 mice/group; values for 1:3000 dilution of sera presented.             

These experiments indicate that the mice immunized with pCOC12 or pBGC23proteins that went through the renaturation protocol raised antibodiesthat cross-react with the purified E. tenella TA4 antigen. These seragave a strong positive signal with the purified E. tenella TA4 antigento at least a 1:3000 dilution. On the other hand, sera from miceinjected with insoluble pCOC12 and pBGC23 proteins had essentially nocross-reacting antibodies to the E. tenella TA4 antigen even at seradilutions as low as 1:30. These experiments indicate that theunpurified, non-renatured insoluble pCOC12 and pBGC23 proteins were noteffective immunogens.

EXAMPLE 18 Use of Bacterially Produced TA4 Proteins to Elicit SporozoiteNeutralizing Serum Response and Protective Response Against E. tenellain Chickens

It has been previously demonstrated that administration of the TA4antigen purified from E. tenella (15 micrograms) produced serumantibodies that neutralized sporozoites in vitro and protected chickensagainst an E. tenella challenge. The renatured pCOC12 and pBGC23proteins were tested for both these properties. Beta-galactosidase andrenatured prochymosin were used as controls. Renatured pBGC23 protein,pCOC12 protein, and prochymosin were concentrated by dialysis againstpolyethylene glycol or by hollow fiber filtration (cartridge H1P10-20,Amicon Corp. Danvers, Mass.) to a final concentration of 0.5-2.0 mg/ml.Each antigen was formulated as one volume of protein concentrate tothree volumes of oil carrier consisting of 5% Arlacel, 94% Drakeol 6-VRand 1% Tween 80. The dose of each antigen employed is listed in TableVII. The doses chosen contained approximately 0.5-2 times the molaramount of purified E. tenella native TA4 antigen previously shown to beeffective in evoking an immune response.

                  TABLE VII                                                       ______________________________________                                        ANTIGEN           MICROGRAMS/DOSE                                             ______________________________________                                        Beta-galactosidase                                                                              133                                                         Renatured pBGC23 Protein                                                                        80                                                          Renatured pBGC23 Protein                                                                        160                                                         Renatured Prochymosin                                                                           53                                                          Renatured pCOC12 Protein                                                                        80                                                          ______________________________________                                    

In experiment 1, chickens received 0.2-0.55 cc of the appropriatelyformulated vaccine by intramuscular injection in the neck. Chickensreceived booster vaccinations by the same route two additional timesseparated by two-week intervals. In experiment 2, chickens received0.2-0.45 cc of the appropriately formulated vaccine by injection intoduodenal tissue. Chickens received one booster vaccination by the sameroute two weeks later. Three days prior to each administration ofprotein and eleven days after the final administration birds were bledfor collection of serum samples.

Eliciting Sporozoite Neutralizing Serum Response Against E. tenella

Heat-inactivated sera from chickens in Experiments 1 and 2 were testedfor neutralization of E. tenella sporozoites. The microneutralizationassay was performed with primary chick kidney cell cultures as follows.One to two-week old chicks were sacrificed and ascepticallynephrectomized. The kidneys were trypsinized, and cells plated into 96well cultures at a density of approximately 10⁴ /well in Earles LAHmedium supplemented with 5% heat-inactivated fetal calf serum. Cultureswere maintained at 41° C. in a 5% CO₂ atmosphere. When cell culturesreached a level of approximately 50% confluency, 50 microliters ofappropriately diluted test serum was added to each well of the plate.Next, 2-3×10⁴ sporozoites in 50 microliters of Earles culture mediumwere added to all wells of the plate. Twelve to sixteen hours later, theculture supernatant was replaced with fresh Earle LAH containing 2% heatinactivated fetal calf serum. The cultures were terminated at 40-44hours post-infection. Culture supernatants were emptied from the platesat that time. Subsequently, cells were fixed to the plates by theaddition of methanol, acidified with 5% glacial acetic acid. The fixedcultures were stained with 0.1% toluidine blue before examining. Wellswere scored as to the approximate percentage of inhibition ofschizogony. Neutralization of parasites was scored on the basis of themaximum serum dilution still producing complete inhibition of schizontdevelopment.

The results in Table VIII indicate that whereas birds vaccinated withbeta-galactosidase or renatured prochymosin had no demonstrableneutralizing antiserum titers against E. tenella sporozoites, birdsreceiving three doses of pBGC23 protein or pCOC12 proteinintramuscularly had demonstrable neutralizing antiserum titers.

                  TABLE VIII                                                      ______________________________________                                                                Geometric Mean                                                                Sporozoite                                                                    Neutralizing                                                  Serum Sample    Titers                                                ______________________________________                                        Experiment 1:                                                                           Pre-bleed IM      1:2.0                                                       Adjuvant Only     1:2.0                                                       Beta-galactosidase                                                                              1:2.0                                                       Renatured pBGC23 Protein                                                                        1:3.2                                                       (80 micrograms)                                                               Renatured pBGC23 Protein                                                                        1:2.6                                                       (160 micrograms)                                                              Renatured Prochymosin                                                                           1:2.0                                                       Renatured pCOC12 Protein                                                                        1:4.0                                                       Sporozoite Immune  1:16.0                                           ______________________________________                                    

Demonstration that Neutralizing Serum of Chickens Immunized with E.coli-Produced TA4 Protein Compete with Monoclonal Antibody Ptn 7.2 A4/4

Sera from vaccinated birds with demonstrable neutralization titers to E.tenella sporozoites, as well as corresponding control sera were testedfor the ability to compete with antibody Ptn 7.2 A4/4 for binding siteson sporozoite membranes. Polystyrene 96 well plates (Immulon II) wereincubated with 50 microliters of sporozoite membrane proteins in 10 mMglycine buffered saline, pH 9.6, at a level of approximately 100micrograms total protein/ml overnight at 37° C. After washing platesthree times with PBS-Tween (0.05% Tween-20) plates were incubated for 1hour with 3% (w/v) bovine serum albumin (RIA grade, Sigma Chemical Co.,St. Louis, Mo.) in PBS. Serial two-fold dilutions of sera from 1:2 to1:200 were prepared in 0.15M phosphate buffered saline with 0.0005%Tween-20 and incubated with the plates for 3 hours at 37° C. Plates werethen incubated with alkaline phosphatase conjugated Ptn 7.2 A4/4monoclonal antibody for 1 hour at 37 ° C. The plates were rinsed free ofunreacted materials using 0.15M phosphate buffered saline with (0.0005%)Tween-20. Afterward, 100 microliters of substrate solution consisting of1 mg/ml sodium p-phosphonitrophenol in 1M diethanolamine buffer wasadded to each well. The resultant reaction product was monitoredspectrophometrically. Sera from birds responding to the parenteralvaccination program, as evidenced by neutralization of sporozoites,contained antibody which competed with monoclonal antibody Ptn 7.2 A4/4(Table IX). This experiment provided direct evidence that renaturedpBGC23 and pCOC12 proteins were capable of stimulating an immuneresponse in chickens to a region of the TA4 antigen recognized bymonoclonal antibody Ptn 7.2 A4/4.

                  TABLE IX                                                        ______________________________________                                        Ptn 7.2 A4/4 Competition titers (50% Inhibition)                                                    Reciprocal Titer                                        ______________________________________                                        Experiment 1:                                                                           Pre-bleed         0                                                           Beta-galactosidase                                                                              0                                                           Renatured pBGC23 Protein                                                                        6.5                                                         (80 micrograms)                                                               Renatured pBGC23 Protein                                                                        6.5                                                         (160 micrograms)                                                              Renatured Prochymosin                                                                           0.6                                                         Renatured pCOC12 Protein                                                                        13.1                                                        (80 micrograms)                                                               Renatured pCOC12 Protein                                                                        9.9                                                         (40 micrograms)                                                               Native TA4        14.6                                              ______________________________________                                    

Immunization with Various TA4 Proteins Reduced the Severity of Infectionin Chickens Challenged with E. tenella

Eleven days after the last vaccination, chickens were challenged with alow level of coccidia (ca. 300-500 oocysts) and maintained in floorpens. The bedding was not removed so as to maximize oocyst recycling.Chickens received a second challenge of 4000-5000 oocysts one week afterthe primary challenge to maximize uniformity of lesion development.Chickens were sacrificed 6 days later for evaluation of lesiondevelopment. Lesion scores were assessed by the parameters establishedby Johnson and Reid (30).

The results in Table X demonstrate that birds vaccinated with renaturedpBGC23 or pCOC12 protein developed less severe lesions followingchallenge than did the corresponding control groups. Vaccination witheither renatured pBGC23 or pCOC12 protein not only abolished thedevelopment of the most severe lesions (level=4) but also shifted thedistribution of lesion severity to lower values. Approximately 50-70% ofvaccinated birds registered lesions of 1-2 whereas 50-70% of the controlbirds had lesion scores of 3-4.

                  TABLE X                                                         ______________________________________                                                    % Distribution Lesion Scored                                      Treatment     0      1       2     3     4                                    ______________________________________                                        Experiment 1                                                                  Beta-galactosidase                                                                          0      0       22.2  50.0  27.8                                 Renatured pBGC23                                                                            0      13.8    38.5  61.5  0                                    Protein (80 micro-                                                            grams)                                                                        Renatured pBGC23                                                                            0      30.8    38.5  30.8  0                                    Protein (160 micro-                                                           grams)                                                                        Renatured Prochy-                                                                           0      7.1     21.4  57.1  14.3                                 mosin                                                                         Renatured pCOC12                                                                            0      11.1    44.4  44.4  0                                    Protein                                                                       Nonvaccinated Con-                                                                          0      0       12.5  68.5  18.8                                 trol                                                                          Experiment 2                                                                  Beta-galactosidase                                                                          0      8       27    37    28                                   Renatured pBGC23                                                                            0      34      44    22    0                                    Protein (160 micro-                                                           grams)                                                                        Renatured Prochy-                                                                           0      0       29    29    42                                   mosin                                                                         Renatured pCOC12                                                                            0      42      14    14    30                                   Protein                                                                       Nonvaccinated Con-                                                                          0      0       0     20    80                                   trol                                                                          ______________________________________                                    

EXAMPLE 19 Response In Chickens To Exposure To Direct ExpressionProduced Recobminant Ta4(pDET) Antigen

Serologic response of pDET vaccinated chickens to Eimeria tenellaantigen. Experiments were conducted to demonstrate the immunoreactivityof pDET vaccinated chickens to the sporocyst derived membrane protein ofEimeria tenella. In one experiment, ten birds were vaccinated with 50micrograms of renatured pDET antigen, a direct expression of producedprotein, production of which is referred to in Example 10 and 13.Immunoreactivity of the protein was assayed and confirmed with themonoclonal antibody Ptn 7.2A4/4 prior to its incorporation in theexperiment.

Vaccine was prepared using a 3:1 ratio of 5% Arlacel-A, 94% Drakeol 6-VRand 1% Tween 80 to pDET with 0.04 micrograms of LPS (three-dose study)or 50 micrograms PHA (one-dose study), and administered in asubcutaneous 0.5 ml dose in the neck behind the head. In one experimentstarting with 2-week-old Leghorns, the vaccination regimen consisted ofthree doses at 10-day intervals. Birds were bled at this same intervaland serum was collected and stored frozen. The second experiment used4-day-old broilers, which were bled 5 days after vaccination. Controlsfor both experiments consisted of an inactive insoluble pDET adjuvant inthe above carrier; adjuvant/carrier; and nonvaccinated controls.

Sera from the vaccinated and controls were analyzed for immunereactivity using Western blot against Eimeria tenella sporocyst proteinas described in Example 13.

The results set forth in Table XI below indicate that 9 of 10 birds inthe 3-dose study vaccinated with pDET antigen responded with a positivereaction at the appropriate molecular weight band 10 days after theinitial exposure, with 10 of 10 reacting after the two subsequentexposures. After the second exposure to insoluble pDET, several birdsbecame seropositive to TA4 antigen. In the one-dose experiment, 10 of 10birds vaccinated with pDET seroconverted on Western blot analysis after5 days. None of the LPS or challenge control birds became seropositivethroughtout either test.

                  TABLE XI                                                        ______________________________________                                        pDET Antigen Immunoreactivity Assay for Three                                 and One-Dose Exposures                                                        Vaccine                                                                       Group        1st Bleed 2nd Bleed  3rd Bleed                                   ______________________________________                                        pDET         9/10      10/10      10/10                                       Antigen (n = 10)                                                              Insoluble    0/10      8/10       10/10                                       pDET (n = 10)                                                                 Adjuvant     0/10      0/10        0/10                                       Control (n = 10)                                                              Nonvaccinate 0/10      0/10        0/10                                       Control (n = 10)                                                              pDET (One Dose)                                                                            10/10     --         --                                          Antigen (n = 10)                                                              Adjuvant Control                                                                           0/10      --         --                                          (One Dose)                                                                    (n = 10)                                                                      ______________________________________                                    

Protection of pDET vaccinated chickens from challenge with Eimeriatenella oocysts. Ten days following the 3-dose vaccination scheduleoutlined above, chickens were inoculated with Eimera tenella oocysts,and examined for pathognomonic lesions of the parasite. The four groupsmentioned above (pDET antigen, insoluble pDET, adjuvant control,nonvaccinate controls) were exposed per Os to 6,000 sporulated oocysts10 days after final vaccination. The inoculum had been previouslytitrated to result in the desired severity of lesion. Caecal lesionscharacteristic for the parasite were scored five days after challenge,as in Example 18. The results as shown in Table XII below demonstrate areduction in severity of lesions, and decrease in mortality in the pDETantigen group as compared to the controls.

                  TABLE XII                                                       ______________________________________                                        Lesions Scores of pDET Vaccinated Chickens When                               Challenged with Eimeria Tenella Oocysts                                       Treatment      Lesion Score X + s.d.                                                                         # Deaths                                       ______________________________________                                        pDET Antigen (n = 10)                                                                        3.2 ± 0.4    0                                              Insoluble pDET (n = 10)                                                                      3.4 ± 0.5    1                                              Adjuvant Controls                                                                            3.8 ± 0.4    3                                              (n = 10)                                                                      Nonvaccinate Controls                                                                        3.9 ± 0.3    3                                              (n = 10)                                                                      ______________________________________                                    

Sporozoite neutralizing serum response in chickens vaccinated with pDETTA4 antigen. A sporozoite neutralization assay (SNA) was utilized toassess the ability of pDET to confer parasite neutralizing capacity tothe serum of vaccinated birds. Using the SNA protocol extablished inExample 18, the sera from the above mentioned one and three doseexperiments were assayed for neutralizing sera.

As shown in Table XIII below, the pDET vaccinated birds demonstratedsporozoite neurtralizing capability conferred to their sera whencompared with the proper controls.

                  TABLE XIII                                                      ______________________________________                                        Sporozoitye Neutralization Assay for                                          pDET Vaccinated Birds                                                         Treatment                                                                     Group        <1:4    1:4     1:16  1:18  1:32                                 ______________________________________                                        pDET Antigen 0/8     1/8     3/8   4/8   --                                   (3 dose n = 8)                                                                Insoluble pDET                                                                             5/8     1/8     2/8   --    --                                   (3 dose n = 8)                                                                Adjuvant     6/8     2/8     --    --    --                                   Control                                                                       (3 dose n = 8)                                                                E. tenella   --      --      --    --    2/2                                  Sporozoite                                                                    Immune Sera                                                                   (n = 2)                                                                       pDET Antigen --      --      2/4   2/4   --                                   (1 dose n = 4)                                                                Adjuvant     5/5     --      --    --    --                                   Control                                                                       (1 dose n = 5)                                                                ______________________________________                                    

EXAMPLE 20

A vaccine for immunization of chickens against coccidiosis and otherdisease agents may be prepared from the genetically engineered E.tenella TA4 sporozoite membrane protein and an avian viral vaccineantigen, namely Infectious Bursal Disease Virus. A suitable carrier forthis combination of antigens is 5% Arlacel A, 94% Drakeol 6-VR, 1%Tween-80. The vaccine may be prepared by formulating one part of anaqueous solution of antigen with 3 parts Arlacel A/Drakeol 6-VR to afinal concentration of 10 to 200 micrograms of each antigen/dose. Thevaccine may be administered to chickens of any age by any route, forexample by the intramuscular route. Properly vaccinated birds areprotected against disease, (including depressed performance or death),caused by field challenge with the species contained in the vaccinewhich includes at least one Eimeria antigen epitope.

EXAMPLE 21 Response Of Chickens To A Multi-Component Exposure OfRecombinant Eimeria Tenella (TA4) Antigen and Recombinant Eimeria MaximaAntigen

An experiment was conducted to demonstrate the immunoreactivity of birdsvaccinated with both E. tenalla (pCOC20) and E. maxima recombinantantigens (p5-3, p14-9, or p11-2). Birds were divided into groupsreceiving the following:

    ______________________________________                                        Treatment                                                                     Group #    Antigen      Quantity of Antigen                                   ______________________________________                                        I          pCOC20       50 micrograms                                         II         p5-3         50 micrograms                                         III        p14-9        50 micrograms                                         IV         p11-2        50 micrograms                                         V          pCOC20 & p5-3                                                                              100 micrograms                                                                (50 micrograms each)                                  VI         pCOC20 & p14-9                                                                             100 micrograms                                                                (50 micrograms each)                                  VII        p5-3 & p14-9 50 micrograms                                                                 (25 micrograms each)                                  VIII       Adjuvant Control                                                                           --                                                    IX         Unvaccinated --                                                    ______________________________________                                    

Vaccines were formulated as described in Examples 42 and 43 using a 3:1(v/v) ratio of carrier to antigen, with the addition of Salmonellaminnesota LPS to a final concentration of 8 micrograms/ml, to a totalantigen concentration of 100 micrograms or 200 micrograms per ml. Thisformulation was delivered as a 0.5 ml subcutaneous dose behind the head.Vaccination regimen began with 2-week leghorns which received 3 doses at10-day intervals, with birds bled, and sera collected and stored frozenafter each vaccination. Controls for the experiment consisted ofcarrier/LPS and nonvaccinated challenge birds.

Sera from the vaccinates and controls were assessed for immunereactivity against both E. tenella sporocyst derived membrane proteinand E. maxima whole merozoite protein by Western blot analysis and byindirect fluorescent antibody staining.

Ten days following the last vaccination, all groups were initiallyinoculated per os with 500 E. tenella and/or 100 E. maxima infectiveoocysts followed five days later by a second challenge of 4000 E.tenella and 40,000 E. maxima infective oocysts. Cecal lesions of E.tenella and duodenal lesions of E. maxima, both pathognomonic for theirrespective pathogens, were scored five days after the second challenge.

Parasite neutralization assays were employed to assess the serumresponse of birds vaccinated with a combination of E. tenella and E.maxima recombinant antigens. Activity against E. maxima was assessedusing an in vivo neutralization assay described in Example 33. Theresults are tabulated in Tables XXIV, XXV, XXVI and XXVII below.

                  TABLE XXIV                                                      ______________________________________                                        Lesion scored of recombinant TA4 and recombinant 8B5                          immunoreactive antigen vaccinated birds when challenged                       with E. maxima oocysts (40,000)*                                              Treatment     E. maxima                                                       Group         Lesion Scores                                                   ______________________________________                                        I              2.2 ± 0.62                                                  III           2.56 ± 0.42                                                  IV             2.5 ± 0.42                                                  VII           2.39 ± 0.42                                                  I              2.8 ± 0.27                                                  VIII          2.75 ± 0.35                                                  IX            2.75 ± 0.38                                                  ______________________________________                                    

                  TABLE XXV                                                       ______________________________________                                        Lesion scores of recombinant TA4 and recombinant 8B5                          immunoreactive antigen vaccinated birds when challenged                       with E. tenella (4,000) and E. maxima oocysts (40,000)*                       Treatment     E. tenella E. maxima                                            Group #       Lesion Scores                                                                            Lesion Scores                                        ______________________________________                                        VI            2.56 ± 0.82                                                                           2.37 ± 0.58                                       V             2.83 ± 0.66                                                                           2.28 ± 0.36                                       VIII          3.75 ± 0.35                                                                           3.5 ± 0                                           IX            2.87 ± 0.63                                                                            2.5 ± 0.71                                       ______________________________________                                    

                  TABLE XXVI                                                      ______________________________________                                        Lesion scores of recombinant TA4 and recombinant 8B5                          immunoreactive antigen vaccinated birds when challenged                       with E. tenella oocyts (4,000)*                                               Treatment     E. tenella                                                      Group #       Lesion Scores                                                   ______________________________________                                        I             2.5 ± 0.84                                                   VIII          3.5 ± 0.5                                                    IX            3.0 ± 1.08                                                   ______________________________________                                         *Birds received a prechallenge with 100 E. maxima oocysts, 500 E. tenella     oocysts or both 4 days prior to heavy challenge.                         

                  TABLE XXVII                                                     ______________________________________                                        In vivo E. maxima merozoite neutralization assay for                          renatured recombinant 8B5 immunoreactive antigen and                          recombinant TA4 antigen vaccinated birds.                                     Treatment  Oocyst Ouput                                                                              Mean Oocyst                                            Group #    % of Adjuvant                                                                             Output/Bird (×10.sup.6)                          ______________________________________                                        II         15          23                                                     II         26          38                                                     III        18          27                                                     IV         77          115                                                    V          38          58                                                     V           0           0                                                     VII        69          104                                                    VI         21          31                                                     I          67          100                                                    VIII       100         150                                                    IX*         0           0                                                     ______________________________________                                         * = unchallenged                                                         

EXAMPLE 22 Weight Gain In Chickens Following Vaccination with pCOC20

Three different lots of pCOC20 (TA4) antigen were evaluated in a singledose vaccination/challenge study. Broilers were inoculatedsubcutaneously behind the neck with the antigen (100 micrograms)adjuvanted with PHA (50 micrograms). Vaccinations were at 5-6 days ofage. Fourteen days later, the birds were bled, weighed, bile collectedfrom selected birds, and inoculated per os with 5,000 sporulated E.tenella oocysts. The inoculum had been previously titrated to result inthe desired severity of lesion. Six days after challenge, the birds werebled, reweighed and several birds were killed and lesions scored. Theremaining birds were weighed again at 10 days after challenge. Lesionscores of vaccinated birds were not significantly different from thoseof nonvaccinated birds. Weight gains are presented in Table XXVIIIbelow.

                  TABLE XXVIII                                                    ______________________________________                                        Weight Performance in pCOC20                                                  Vaccinated Birds Following E. tenella Challenge                                             Percent Weight Gain .sup.--X ± s.d.                                        Days Post-Challenge                                             Treatment Group 0-6       6-10     0-10                                       ______________________________________                                        Nonchallenged (n = 19)                                                                        52 ± 5 29 ± 8                                                                              96 ± 10                                 Nonvaccinated (n = 19)                                                                        39 ± 6 28 ± 8                                                                              78 ± 10                                 Adjuvant (n = 20)                                                                             39 ± 6 28 ± 7                                                                              77 ± 9                                  Chymosin (n = 19)                                                                             40 ± 8 28 ± 7                                                                              79 ± 13                                 pCOC20 (n = 17) 41 ± 5 32 ± 5                                                                              86 ± 10                                 pCOC20 (n = 16)  43 ± 10                                                                             32 ± 8                                                                              88 ± 15                                 pCOC20 (n = 15) 41 ± 6 35 ± 9                                                                              90 ± 11                                 pDET (n = 13)   43 ± 3 35 ± 7                                                                              93 ± 11                                 ______________________________________                                    

The percent weight gains over the 10-day challenge period indicate thatvaccination with pCOC20 provided a degree of protection upon challenge.The chymosin/adjuvant and adjuvant only groups were no different thanthe nonvaccinated challenged group.

EXAMPLE 23 Vaccination/Challenge Weight Performance Trial pCOC20 Vs. E.tenella

A total of 144 birds (8 days of age) were divided into four groups of 36birds each. The first group of birds was vaccinated subcutaneously with0.5 ml of a water in oil emulsion containing 100 μg of pCOC20 antigenand 50 μg of PHA. The second group of birds received the same emulsionwithout antigen. The third and fourth groups of birds served asnonvaccinate controls. Birds were revaccinated at 18 days of age with0.25 ml of emulsion containing 50 μg of pCOC20 antigen and 25 μg of PHA.

At 22 days of age, the first three groups of birds were challenged with10,000 E. tenella oocysts followed two days later by 70,000 additionaloocysts. The fourth group of birds were housed in a separate pen andremained unchallenged.

The birds were weighed 6, 8, 10 and 14 days after the initial challenge.The data was analyzed by the Duncan multiple range test for statisticalsignificance. The data as well as the statistical analysis appear inTable XXIX below and FIG. 25.

The results reveal that the vaccinate group out performed the otherchallenged control groups. At 14 days post-challenge, the vaccinategroup was not significantly different from the control nonchallengedgroup and yet significantly heavier than the nonvaccinate challengecontrol birds. The birds receiving adjuvant only were significantlylighter than the nonchallenged controls but not significantly differentfrom the nonvaccinate challenge control group. These results clearlydemonstrate the protective nature of the pCOC20 antigen.

                  TABLE XXIX                                                      ______________________________________                                        STATISTICAL ANALYSIS OF INDIVIDUAL BIRD                                       WEIGHTS DUNCAN M.R.T. WITH 95% C.I.                                                       Days Post-challenge                                               Treatment Group                                                                             0      6       8     10    14                                   ______________________________________                                        VAC/CH vs CTL/CH                                                                            NS     NS      S     S     S                                    VAC/CH vs CTL/NC                                                                            NS     S       S     S     NS                                   CTL/CH vs CTL/NC                                                                            NS     S       S     S     S                                    CTL/CH vs ADJ/CH                                                                            NS     NS      NS    NS    NS                                   ADJ/CH vw CTL/NC                                                                            NS     S       S     S     S                                    ______________________________________                                         S = Significantly Different                                                   NS = Not Significantly different                                         

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What is claimed is:
 1. An isolated nucleic acid molecule encoding a17,000 dalton antigenic protein derived from Eimeria tenella and havingthe amino acid sequence shown in FIG.
 1. 2. The nucleic acid molecule ofclaim 1, wherein said molecule is an mRNA molecule.
 3. The nucleic acidmolecule of claim 1, wherein said molecule is a cDNA molecule.
 4. Anisolated nucleic acid molecule ending a 8,000 dalton antigenic proteinderived from Eimeria tenella and having the amino acid sequence asfollows: AlaAlaGlythrThrAspAlaValIleCysLeuThrAsnProAlaProLeuGluAlaArgSerGlnProPheAspAspGluGlnTrpLysLysIleValAspSerLeuSerLeuSerGluGluGluGluGluLysGlyGlyValSerProValValProSerValAlaLeuIleSerAlaAlaValIleSerAlaPheAlaLeuPhe.
 5. The nucleic acidmolecule of claim 4, wherein said molecule is an mRNA molecule.
 6. Thenucleic acid molecule of claim 4, wherein said molecule is a cDNAmolecule.
 7. A recombinant cloning vector which comprises the isolatednucleic acid molecule of claim
 5. 8. A recombinant cloning vector whichcomprises the isolated nucleic acid molecule of claim
 4. 9. A bacterialhose cell which comprises the recombinant cloning vector of claim 7 or8.
 10. An expression vector which comprises the cDNA molecule of claim 3operably linked to a promoter of DNA expression.
 11. An expressionvector which comprises the cDNA molecule of claim 6 operably linked to apromoter of DNA expression.
 12. An expression vector which comprises apromoter of DNA expression operably linked to the 5' end of DNA encodingβ-galactosidase, the 3' end of which DNA is directly linked to the 5'end of the cDNA molecule of claim
 3. 13. An expression vector whichcomprises a promoter of DNA expression operably linked to the 5' end ofDNA encoding β-galactosidase, the 3' end of which DNA is directly linkedto the 5' end of the cDNA molecule of claim
 6. 14. An expression vectorwhich comprises a promoter of DNA expression operably linked to the 5'end of DNA encoding β-galactosidase, the 3' end of which DNA is directlylinked to the 5' end of the cDNA molecule of claim
 3. 15. The expressionvector of claim 14, wherein the DNA encoding prochymosin contains a 249base pair deletion.
 16. An expression vector which comprises a promoterof DNA expression operably linked to the 5' end of DNA encodingβ-galactosidase, the 3' end of which DNA is directly linked to the 5'end of the cDNA molecule of claim
 6. 17. The expression vector of claim16, wherein the DNA encoding prochymosin contains a 249 base pairdeletion.
 18. A bacterial host cell which comprises the expressionvector as in any one of claims 10-17.
 19. A method of preparing anantigenic protein derived from Eimeria tenella which comprises growingthe bacterial hose cell of claim 18 under conditions permitting DNAexpression and protein production followed by recovering the protein soproduced.
 20. The method of claim 19, wherein the recovery comprises:(i)separating the protein from the host cells; (ii) purifying the protein;(iii) solubilizing the protein; (iv) renaturing the protein; and (v)recovering the purified, solubilized and renatured protein.